Novel anti-glypican 3 antibody and pharmaceutical composition containing the same

ABSTRACT

The present invention relates to an antibody specifically binding to glypican 3 (GPC3), a nucleic acid encoding the antibody, a vector and a host cell containing the nucleic acid, a method of preparing the antibody, and a pharmaceutical composition for treating cancer or tumor, containing the antibody as an active ingredient. The antibody specifically binding to glypican 3 according to the present invention may be effectively used to treat cancer or tumor, particularly, hepatocellular carcinoma due to high affinity and specificity to glypican 3.

TECHNICAL FIELD

The present invention relates to an antibody specifically binding toglypican 3 (GPC3), a nucleic acid encoding the antibody, a vector and ahost cell containing the nucleic acid, a method of preparing theantibody, and a pharmaceutical composition for treating cancer or tumor,containing the antibody as an active ingredient.

BACKGROUND ART

Glypican 3 was separated in the small intestine of a rat as a transcriptin a developmental stage (Mol. Cell Biol. 8, 4243-4249, 1988), andthereafter, it was reported that a gene encoding glypican 3 was isolatedfrom a glycosyl-phosphatidylinositol (GPI)-linked type sulfate humangastric cancer cell line having a core protein of a glypican family(molecular weight of 69 kDa) (Gene, 188, 151-156, 1997). It was reportedthat glypican 3 forms a protein-protein complex together with aninsulin-like growth factor-2 and regulates actions of the insulin-likegrowth factor-2 (Nat. Genet., 12, 241-247, 1996). Particularly, it wasknown that glypican 3 is expressed in the fetal liver and placentaduring development and in normal adult tissue, expression of glypican 3is deteriorated or glypican 3 is not expressed at all.

Glypican 3 is known as a kind of oncofetal antigen that belongs to aglypican family of glycosyl-phosphatidylinositol (GPI)-anchored heparinsulfate proteoglycans, and cell membrane-bound glypican 3 is known to becomposed of two subunits linked by one or more disulfide bonds.

In relation to functions and uses of glypican 3, it was reported thatglypican may serve as a hepatocellular carcinoma marker, and it wassuggested that glypican may serve as a receptor of endostatin having apossibility of an angiogenesis inhibitor, which was not clearlyidentified.

Recently, it has been known that glypican 3 is expressed in variouscancers, particularly, hepatocellular carcinoma (HCC), melanoma, Wilm'stumor, and hepatoblastoma (Jakubovic and Jothy; Ex. Mol. Path.82:184-189 (2007); Nakatsura and Nishimura, Biodrugs 19(2):71-77(2005)), and it was reported that it is actually possible to treattumors such as hepatic cancer, and the like, using an antibody againstglypican 3 (Korean Patent No. 877,176, and the like).

Currently, several anti-glypican 3 antibodies were reported, but anantibody having a satisfactory therapeutic effect, particularly, anexcellent cancer therapeutic effect has been not yet reported.Therefore, a need for an anti-glypican 3 antibody having a moreexcellent therapeutic effect is significant.

Therefore, the present inventors invented a novel antibody specificallybinding to glypican 3 with high affinity, and confirmed a possibility ofthe antibody according to the present invention as an efficientanti-cancer agent, thereby completing the present invention.

DISCLOSURE

An object of the present invention is to provide a novel antibodyspecifically binding to glypican 3 (GPC3) with high affinity, a nucleicacid encoding the antibody, a vector and a host cell containing thenucleic acid, a method of preparing the antibody, and a pharmaceuticalcomposition for treating cancer or tumor, containing the antibody as anactive ingredient.

According to an aspect of the present invention, there is provided anovel antibody specifically binding to glypican 3 (GPC3) with highaffinity.

According to another aspect of the present invention, there are provideda nucleic acid encoding an anti-glypican 3 antibody according to thepresent invention; a vector containing the nucleic acid; and a celltransduced with the vector, and a method of preparing an antibodyaccording to the present invention, using the same.

According to another aspect of the present invention, there are provideda pharmaceutical composition for treating cancer or tumor, containing ananti-glypican 3 antibody according to the present invention as an activeingredient, and a method of treating cancer or tumor using theanti-glypican 3 antibody according to the present invention.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a view illustrating a result of confirming expression of GPC3and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in each cell line.

FIG. 2 is a view illustrating a structure of a plasmid used forexpression of GPC3 in each cell line.

FIG. 3 is a view illustrating that a cell line colony transformed so asto express GPC3 using resistance against Hygromycin may be confirmed.

FIG. 4 is a view illustrating a result of analyzing an expression amountof GPC3 in each cell line.

FIG. 5 is a view illustrating that an anti-glypican 3 antibody binds toa cell line transformed so as to express GPC3.

FIG. 6 is a view illustrating binding affinity of an anti-glypican 3antibody according to the present invention to a transformed cell line(SK-Hep1-GPC3) so as to express GPC3.

FIG. 7 is a view illustrating binding affinity of the anti-glypican 3antibody according to the present invention to a cell line (HepG2)expressing GPC3.

FIG. 8 is a view illustrating binding affinity of the anti-glypican 3antibody according to the present invention to a cell line (HepG2)expressing GPC3.

FIG. 9 is a view illustrating results obtained by confirming whether ornot GX102, GX270, and GS012 clone antibodies according to the presentinvention selectively bind to cell lines in proportion to an expressionlevel of GPC3 in seven types of hepatic cancer cell lines (SK-Hep1,PLC/PRF/5, SNU398, Hep3B, Huh7, HepG2, and SK-Hep1-GPC3 #9) and one kindof normal hepatic cancer cell line (CHANG).

(a) SKHep1 (GPC3 negative cell line)

(b) SKHep1-GPC3 #9 (GPC3 positive cell line)

FIG. 10 is a view illustrating results obtained by confirming whether ornot the GX102, GX270, and GS012 clone antibodies according to thepresent invention selectively bind to cell lines in proportion to anexpression level of GPC3 in seven kinds of hepatic cancer cell lines(SK-Hep1, PLC/PRF/5, SNU398, Hep3B, Huh7, HepG2, and SK-Hep1-GPC3 #9)and one kind of normal hepatic cancer cell line (CHANG), representingand comparing the results by positive signs depending on an overlappingprofile of fluorescence-activated cell sorting (FACS) histogram withthat in an isotope negative control group.

DETAILED DESCRIPTION OF THE EMBODIMENTS

As used herein, the term “glypican 3” or “GPC3” collectively indicatesarbitrary mutants, isomers, and homologues of glypican 3 as well as GPC3present in animal, preferably, human bodies, as it is.

As used herein, the term “human GPC3” means GPC3 of a human andpreferably has an amino acid sequence (SEQ ID NO: 393) of Genbankaccession number AAH35972.1, but is not limited thereto.

The present invention provides an anti-glypican 3 antibody having aheavy chain variable region including a heavy chain CDR1 comprising anamino acid sequence of SEQ ID NO: 133, 143, 149, 159, 169, 174, 184,199, 262, 268, 273, 281, 285, 291, 297, 308, 353, 363, 374, 380, or 386;

-   -   a heavy chain CDR2 comprising an amino acid sequence of SEQ ID        NO:134, 144, 150, 160, 170, 175, 185, 200, 231, 263, 266, 269,        274, 282, 286, 292, 298, 303, 309, 314, 318, 323, 328, 331, 336,        341, 345, 349, 354, 359, 364, 369, 375, 381, or 387; and    -   a heavy chain CDR3 comprising an amino acid sequence of SEQ ID        NO: 135, 139, 145, 151, 155, 161, 165, 171, 176, 180, 186, 189,        194, 195, 201, 205, 207, 210, 214, 218, 222, 227, 232, 237, 239,        243, 247, 250, 254, 258, 259, 264, 267, 270, 275, 278, 279, 283,        287, 293, 299, 304, 310, 315, 319, 324, 329, 332, 337, 342, 346,        350, 355, 360, 365, 370, 376, 382, or 388.

In another aspect, the present invention provides an anti-glypican 3antibody having a light chain variable region including a light chainCDR1 comprising an amino acid sequence of SEQ ID NO: 136, 140, 146, 152,156, 162, 166, 172, 177, 181, 187, 190, 193, 196, 202, 208, 211, 215,219, 223, 224, 228, 233, 240, 244, 251, 255, 260, 265, 271, 288, 294,300, 305, 311, 316, 320, 325, 333, 338, 343, 347, 356, 361, 366, 371,377, 383, or 389;

-   -   a light chain CDR2 comprising an amino acid sequence of SEQ ID        NO: 137, 141, 147, 153, 157, 163, 167, 178, 182, 191, 197, 203,        209, 212, 216, 220, 225, 229, 234, 236, 241, 245, 252, 256, 276,        284, 289, 295, 301, 306, 312, 317, 321, 326, 330, 334, 339, 351,        357, 362, 367, 372, 378, 384, 390, or 392; and    -   a light chain CDR3 comprising an amino acid sequence of SEQ ID        NO: 138, 142, 148, 154, 158, 164, 168, 173, 179, 183, 188, 192,        198, 204, 206, 213, 217, 221, 226, 230, 235, 238, 242, 246, 248,        249, 253, 257, 261, 272, 277, 280, 290, 296, 302, 307, 313, 322,        327, 335, 340, 344, 348, 352, 358, 368, 373, 379, 385, or 391.

Preferably, the anti-glypican 3 antibody according to the presentinvention has a heavy chain variable region including a heavy chain CDR1comprising an amino acid sequence of SEQ ID NO: 133, 143, 149, 159, 169,174, 184, 199, 262, 268, 273, 281, 285, 291, 297, 308, 353, 363, 374,380, or 386;

-   -   a heavy chain CDR2 comprising an amino acid sequence of SEQ ID        NO:134, 144, 150, 160, 170, 175, 185, 200, 231, 263, 266, 269,        274, 282, 286, 292, 298, 303, 309, 314, 318, 323, 328, 331, 336,        341, 345, 349, 354, 359, 364, 369, 375, 381, or 387; and    -   a heavy chain CDR3 comprising an amino acid sequence of SEQ ID        NO: 135, 139, 145, 151, 155, 161, 165, 171, 176, 180, 186, 189,        194, 195, 201, 205, 207, 210, 214, 218, 222, 227, 232, 237, 239,        243, 247, 250, 254, 258, 259, 264, 267, 270, 275, 278, 279, 283,        287, 293, 299, 304, 310, 315, 319, 324, 329, 332, 337, 342, 346,        350, 355, 360, 365, 370, 376, 382, or 388, and    -   a light chain variable region including a light chain CDR1        comprising an amino acid sequence of SEQ ID NO: 136, 140, 146,        152, 156, 162, 166, 172, 177, 181, 187, 190, 193, 196, 202, 208,        211, 215, 219, 223, 224, 228, 233, 240, 244, 251, 255, 260, 265,        271, 288, 294, 300, 305, 311, 316, 320, 325, 333, 338, 343, 347,        356, 361, 366, 371, 377, 383, or 389;    -   a light chain CDR2 comprising an amino acid sequence of SEQ ID        NO: 137, 141, 147, 153, 157, 163, 167, 178, 182, 191, 197, 203,        209, 212, 216, 220, 225, 229, 234, 236, 241, 245, 252, 256, 276,        284, 289, 295, 301, 306, 312, 317, 321, 326, 330, 334, 339, 351,        357, 362, 367, 372, 378, 384, 390, or 392; and    -   a light chain CDR3 comprising an amino acid sequence of SEQ ID        NO: 138, 142, 148, 154, 158, 164, 168, 173, 179, 183, 188, 192,        198, 204, 206, 213, 217, 221, 226, 230, 235, 238, 242, 246, 248,        249, 253, 257, 261, 272, 277, 280, 290, 296, 302, 307, 313, 322,        327, 335, 340, 344, 348, 352, 358, 368, 373, 379, 385, or 391.

In another aspect, the anti-glypican 3 antibody according to the presentinvention, contains a heavy chain variable region having a sequence witha sequence homology of 80% or more, preferably 90% or more, morepreferably 95% or more, and most preferably 99% or more with an aminoacid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23,25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59,61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95,97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125,127, 129, or 131, or

-   -   contains a light chain variable region having a sequence with a        sequence homology of 80% or more, preferably 90% or more, more        preferably 95% or more, and most preferably 99% or more with an        amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16,        18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48,        50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80,        82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108,        110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, or 132.

Preferably, the anti-glypican 3 antibody according to the presentinvention

-   -   contains a heavy chain variable region having a sequence with a        sequence homology of 80% or more, preferably 90% or more, more        preferably 95% or more, and most preferably 99% or more with an        amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17,        19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49,        51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81,        83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109,        111, 113, 115, 117, 119, 121, 123, 125, 127, 129, or 131, and    -   a light chain variable region having a sequence with a sequence        homology of 80% or more, preferably 90% or more, more preferably        95% or more, and most preferably 99% or more with an amino acid        sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,        24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54,        56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86,        88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114,        116, 118, 120, 122, 124, 126, 128, 130, or 132.

Further, it is apparent to those skilled in the art that anti-glypican 3antibodies of which some of the amino acids are substituted, inserted,and/or deleted in the heavy chain and light chain variable regions arealso included in the scope of the present invention as long ascharacteristics such as an affinity and specificity to glypican 3, andthe like, satisfying the object of the present invention are maintained.As an example, there is an antibody of which amino acid isconservatively substituted in a variable region. Conservativesubstitution means substitution with another amino acid residue havingsimilar characteristics as those of an original amino acid sequence. Forexample, lysine, arginine, and histidine have similar characteristics inthat they have basic side chains, and aspartic acid and glutamic acidhave similar characteristics in that they have acidic side chains.Further, glycine, asparagines, glutamine, serine, threonine, tyrosine,cysteine, and tryptophane have similar characteristics in that they haveuncharged polar side chains, alanine, valine, leucine, threonine,isoleucine, proline, phenylalanine, and methionine have similarcharacteristics in that they have non-polar side chains, and tyrosine,phenylalanine, tryptophane, and histidine have similar characteristicsin that they have aromatic side chains. Therefore, since it is apparentto those skilled in the art that even though an amino acid issubstituted with another amino acid in an amino acid group havingsimilar characteristics as described above, there is no particularchange in characteristics, an antibody of which mutation is generated byconservative substitution in a variable region is also included in thescope of the present invention as long as the characteristics of theantibody according to the present invention are maintained.

As used herein, the term “antibody”, which is an immunoglobulin moleculeimmunologically reactive with a specific antigen, means a proteinmolecule serving as a receptor specifically recognizing antigen, andincludes a whole antibody thereof and an antibody fragment thereof.

The antibody fragment in the present invention includes a short chainantibody, a diabody, a triabody, a tetrabody, a Fab fragment, a F(ab′)₂fragment, Fd, scFv, a domain antibody, a minibody, a scab, an IgDantibody, an IgE antibody, an IgM antibody, an IgG1 antibody, an IgG2antibody, an IgG3 antibody, an IgG4 antibody, derivatives of an antibodyconstant region, artificial antibodies based on protein scaffolds, andthe like, which have binding affinity to glypican 3, but is not limitedthereto. It is apparent to those skilled in the art that as long asbinding affinity to glypican 3 is maintained, any type of antibodyfragment according to the present invention exhibits the samecharacteristics as those of the antibody according to the presentinvention.

Meanwhile, in another aspect of the present invention, there is providean antibody-drug conjugate (ADC) in which an anti-cancer drug having atumor cell proliferation suppression effect is bound to theanti-glypican 3 antibody according to the present invention.

The drug capable of being used in the antibody-drug conjugate accordingto the present invention includes an arbitrary compound, moiety, orgroup having cytotoxicity or cell proliferation suppression effect, andincludes (i) chemotherapeutic agents serving as a microtubulininhibitor, a mitosis inhibitor, a topoisomerase inhibitor, or a DNAintercalator; (ii) a protein toxin performing an enzymatic function; and(iii) radioisotopes (radionuclides), and the like. One or more of thecompounds may be used as the drug.

A non-restrictive example of the drug as described above may includemaytansinoid, auristatin, dolastatin, trichothecene, CC1065,calicheamicin and other enediyne antibiotics, taxane, anthracycline,methotrexate, adriamycin, vindesine, vinca alkaloids (vincristine,vinblastine, etoposide), doxorubicin, melphalan, mitomycin C,chlorambucil, daunorubicin, daunomycin, etoposide, teniposide,carminomycin, aminopterin, dactinomycin, mitomycins, bleomycins,esperamicins, 5-fluorouracil, melphalan, other nitrogen mustards andstereoisomers, isosteres, analogues, or derivatives thereof,cis-platinum and cis-platinum analogues, enzymes and fragments thereofcorresponding to other intercalating agents, for example, nucleolyticenzymes, antibiotics, and toxins (enzymatically active toxins or smallmolecule toxins of bacterial, fungal, plant or animal origin), variousantitumor or anticancer agents such as cisplatin, CPT-11, doxorubicin,paclitaxel, docetaxel, and the like, but is not limited thereto.Further, an example of the radioisotope (radionuclide) includes 3H, 14C,32P, 35S, 36Cl, 51Cr, 57Co, 58Co, 59Fe, 90Y, 125I, 131I, 186Re, and thelike, but is not limited thereto. A micro RNA (miRNA), a smallinterfering RNA (siRNA), and a small hairpin RNA (shRNA) capable ofsuppressing expression of a specific oncogene may also be used.

It is preferable that the anti-glypican 3 antibody provided in thepresent invention and the drug are conjugated to each other using afunctional group such as a thiol group, or the like, of an amino acidresidue such as lysine or cysteine, in the antibody. In this case, ifnecessary, the anti-glypican 3 antibody and the drug may also beconjugated to each other in a linker-mediated conjugate using agenerally used linker. It is preferable to use a maleimide oriodoacetamide based linker. In the case of conjugating the drug to theantibody or the fragment thereof, it is preferable that the drug isconjugated to a C-terminal site opposite to an antigen binding site soas not to affect binding ability, specificity, and the like, of theantibody or the fragment thereof to glypican 3, and in the case of usingthe whole antibody instead of the fragment thereof, it is alsopreferable to conjugate the drug to an Fc region.

In addition, the present invention provides a chimeric antigen receptor(CAR)-based therapeutic agent containing the anti-glypican 3 antibodyaccording to the present invention. For example, the therapeutic agentmay be preferably a chimeric antigen receptor T-Cell or chimeric antigenreceptor-natural killer (CAR-NK) cell therapeutic agent, but is notlimited thereto.

In another aspect of the present invention, there is provided abispecific antibody containing the anti-glypican 3 antibody according tothe present invention. The bispecific antibody is an antibody capable ofsimultaneously binding to two kinds of antigens, and may be used in ageneral form in which a pair of different heavy chain and light chaincapable of binding to antigens different from each other are linked, andmay also be used in a form of a bispecific single chain antibody inwhich single-chain antibody fragments (scFv) in which VL and VH arelinked to each other by a short linker peptide are linked in a form ofscFv1-scFv2(-Fc), or a bispecific antibody using a BiTE technology byMicromet Inc. (Germany, see http://www.micromet.de).

The bispecific antibody according to the present invention may bepreferably in a form in which the anti-glypican 3 antibody according tothe present invention is bound to an antibody having binding ability toan immune effector cell-specific target molecule, or a fragment thereof.The immune effector cell-specific target molecule may be preferablyselected from TCR/CD3, CD16(FcγRIIIa) CD44, Cd56, CD69, CD64(FcγRI),CD89, and CD11b/CD18(CR3), but is not limited thereto.

Further, the present invention provides a gene encoding the variableregion of the anti-glypican 3 antibody according to the presentinvention and a recombinant vector containing the same. Apolynucleotide, that is, a gene encoding light chain and heavy chainvariable regions of the antibody according to the present invention orthe fragment may be easily derived by those skilled in the art from anamino acid sequence of the anti-glypican 3 antibody provided in thepresent invention.

As used herein, the term “recombinant vector”, which is an expressionvector capable of expressing a target protein in a suitable host cell,means a genetic construct containing an essential regulatory element towhich a genetic insert is operably linked so as to be expressed. Thegene encoding the anti-glypican 3 antibody according to the presentinvention may also be used in a form in which genes are inserted intoseparate vectors or one vector.

In detail, the polynucleotide encoding the amino acid sequence of theanti-glypican 3 antibody according to the present invention may also beused in a form in which genes are inserted into separate vectors,respectively or inserted into one vector, and may be used in a form inwhich polynucleotides encoding the heavy chain and light chain, thevariable regions thereof, or the like, are inserted into separatevectors, respectively or inserted into one vector.

As used herein, the term “operably linked” means that a nucleic acidexpression regulatory sequence and a nucleic acid sequence encoding thetarget protein are functionally linked to each other so as to performgeneral functions. Operable linkage with a recombinant vector may beperformed using a gene recombination technology wellknown in the art,and site-specific DNA cleavage and linkage may be easily performed usingenzymes generally known in the art, or the like.

An expression vector suitable for producing the anti-glypican 3 antibodyaccording to the present invention may include a signal sequence formembrane targeting or secretion in addition to expression regulatoryelements such as a promoter, an initiation codon, a termination codon, apolyadenylation signal, and an enhancer. The initiation codon and thetermination codon may be generally considered as a portion of anucleotide sequence encoding an immunogenic target protein, needs to befunctional in an individual to whom a genetic construct has beenadministered, and must be in frame together with the coding sequence.The promoter may be generally constitutive or inducible. An example ofthe promoter available in prokaryotic cells may include lac, tac, T3,and T7 promoters, but is not limited thereto. An example of the promoteravailable in eukaryotic cells may include a β-actin promoter, promotersfrom human hemoglobin, human muscle creatine, and human metallothioneinas well as a simian virus 40 (SV40) promoter, a mouse mammary tumorvirus (MMTV) promoter, a human immunodeficiency virus (HIV) promotersuch as a HIV Long Terminal Repeat (LTR) promoter, a moloney viruspromoter, a cytomegalovirus (CMV) promoter, an epstein barr virus (EBV)promoter, a rous sarcoma virus (RSV) promoter, but is not limitedthereto.

The expression vector may include a selectable marker for selecting hostcells containing the vector. The selective marker is to select cellstransformed by the vector, and as the selective marker, markersconferring selectable phenotypes such as drug resistance, nutrientrequirement, cytotoxic agent resistance, or expression of surfaceproteins may be used. Since only cells expressing the selectable markersurvive in the environment treated with a selective agent, transformedcells may be selected. Further, in the case of a replicable expressionvector, the vector may include a replication origin, which is a specificnucleic acid sequence that initiates replication.

As the recombinant expression vector for inserting a foreign gene, avector having various shapes such as plasmid, virus, cosmid, and thelike, may be used. The kind of recombinant vector is not particularlylimited as long as it may serve to express the desired gene and producethe desired protein in various host cells such as prokaryotic cells andeukaryotic cells, but a vector capable of mass-producing a foreignprotein having a shape similar to that in a natural state while having apromoter having strong activity and having strong expression ability maybe preferable.

Various expression host/vector combinations may be used to express theanti-glypican 3 antibody. An example of the expression vector suitablefor eukaryotic cells includes an expression regulatory sequence derivedfrom SV40, bovine papilloma virus, adenovirus, adeno-associated virus,cytomegalovirus, and retrovirus, but is not limited thereto. An exampleof the expression vector usable in bacteria host cells includes abacterial plasmid obtained from Escherichia coli such as pET, pRSET,pBluescript, pGEX2T, a pUC vector, col E1, pCR1, pBR322, pMB9, andderivatives thereof; plasmids having a broader host range such as RP4;phage DNAs exemplified as various phage lambda derivatives such asλgt10, λgt11, and NM989; and other DNA phages such as M13,single-stranded filament type DNA phage, and the like. An expressionvector available in yeast cells may be 2° C. plasmid and a derivativethereof. A vector useful for insect cells may be pVL941.

In another aspect, the present invention provides a host celltransformed with the recombinant vector. The recombinant vector isinserted in a host cell to form a transformant. A host cell suitable forthe vector may be a prokaryotic cell such as Escherichia coli, Bacillussubtilis, Streptomyces sp., Pseudomonas sp., Proteus mirabilis, orStaphylococcus sp. In addition, the suitable host cells may beeukaryotic cells, for example, fungal cells such as Aspergillus sp.,yeast cells such as Pichia pastoris, Saccharomyces cerevisiae,Schizosaccharomyces sp., and Neurospora crassa, other lower eukaryoticcells, and cells of higher eukaryotes such as those from insects. Inaddition, the host cell may be derived from plants or mammals.Preferably, as the host cell, monkey kidney cells 7 (COS7), NSO cells,SP2/0, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney(BHK) cells, Madin-Darby canine kidney (MDCK) cells, myeloma cell lines,HuT 78 cells, HEK293 cells, and the like, may be used, but the presentinvention is not limited thereto. Particularly, the CHO cells may bepreferable.

As used herein, the term “transformation into host cells” may includeany method of introducing nucleic acids into organisms, cells, tissues,or organs, and be performed by selecting standard techniques suitablefor the host cell as known in the art. These methods may include anelectroporation method, a protoplast fusion method, a calcium phosphate(CaPO₄) precipitation method, a calcium chloride (CaCl₂)) precipitationmethod, an agitation method using silicon carbide fiber, anagrobacterium-mediated transformation method, and a PEG, dextransulfate, or lipofectamine and a desiccation/inhibition-mediatedtransformation method, but are not limited thereto.

In another aspect, the present invention provides a method of preparingthe anti-glypican 3 antibody according to the present invention byculturing a host cell transformed with the recombinant vector.

A method of preparing a humanized antibody may include preparing arecombinant vector by inserting a nucleotide sequence encoding theanti-glypican 3 antibody according to the present invention into avector; transforming a host cell with the recombinant vector to culturea transformant; and separating and purifying a humanized antibody fromthe cultured transformant.

In detail, the humanized antibody according to the present invention maybe massproduced by culturing the transformant in which the recombinantvector is expressed in a nutrient medium. Here, general medium andculturing conditions may be suitably selected depending on the hostcell. Conditions such as a temperature, pH of the medium, a culturingtime, and the like, may be controlled appropriately for growth of thecells and mass production of proteins at the time of culturing thetransformant.

The anti-glypican 3 antibody produced recombinantly as described abovemay be collected from the medium or cells lysates. In the case of amembrane-binding type anti-glypican 3 antibody, the anti-glypican 3antibody may be isolated from a membrane by using a suitable surfactantsolution (for example, triton-X 100) or enzymatic cleavage. Cells usedfor expressing the humanized antibody may be destructed by variousphysical or chemical means such as freezing-thawing acclimation,sonication, mechanical destruction, or a cell lysing agent, and beseparated and purified by a general biochemical separation technology(Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., ColdSpring Harbor Laboratory Press (1989); Deuscher, M., Guide to ProteinPurification Methods Enzymology, Vol. 182. Academic Press. Inc., SanDiego, Calif. (1990)). As the separation technology, electrophoresis,centrifugation, gel filtration, precipitation, dialysis, chromatography(ion exchange chromatography, affinity chromatography, immuno-adsorptionchromatography, size exclusion chromatography, and the like),isoelectric focusing, and various modification and composite methodsthereof, may be used, but the present invention is not limited thereto.

In another aspect, the present invention provides a composition fortreating cancer, containing the anti-glypican 3 antibody. As usedherein, the term “anti-cancer” includes “prevention” and “treatment”.Here, the term “prevention” means all actions for suppressing ordelaying cancer diseases by administering the composition containing theantibody according to the present invention, and the term “treatment”means all actions for allowing symptoms of cancer to be alleviated or beadvantageously changed by administering the antibody according to thepresent invention.

The kind of cancer or carcinoma capable of being treated by thecomposition according to the present invention is not particularlylimited, and includes both solid cancer and blood cancer. Preferably,the cancer includes all kinds of cancer in which glypican 3 isexpressed, and more preferably, the cancer may be selected from thegroup consisting of hepatic cancer, hepatocellular carcinoma, gastriccancer, breast cancer, lung cancer, ovarian cancer, bronchial cancer,nasopharyngeal cancer, larynx cancer, pancreatic cancer, bladder cancer,colorectal cancer, colon cancer, uterine cervical cancer, brain cancer,prostate cancer, bone cancer, skin cancer, thyroid cancer, parathyroidcancer, kidney cancer, esophageal cancer, biliary tract cancer, testiscancer, rectal cancer, head and neck cancer, cervical spinal cancer,ureteral cancer, osteosarcoma, neuroblastoma, melanoma, fibrosarcoma,rhabdomyosarcoma, astrocytoma, neuroblastoma and glioma. Mostpreferably, the cancer capable of being treated with the compositionaccording to the present invention is hepatic cancer or hepatocellularcarcinoma.

An anti-cancer composition according to the present invention mayfurther contain a pharmaceutically acceptable carrier. In the case of aformulation for oral administration, a binder, a lubricant, adisintegrant, an excipient, a solubilizer, a dispersant, a stabilizer, asuspending agent, a pigment, a flavoring agent, or the like, may beused, in the case of a formulation for injection, a buffering agent, apreservative, a soothing agent, a solubilizer, an isotonic agent, astabilizer, or the like, may be mixed and used, and in the case of aformulation for local administration, a base, an excipient, a lubricant,a preservative, or the like, may be used. The formulation of thepharmaceutical composition according to the present invention may bevariously prepared by mixing the pharmaceutical composition with thepharmaceutically acceptable carrier as described above. For example, inthe case of the formulation for oral administration, the pharmaceuticalcomposition may be prepared in forms of a tablet, a troche, a capsule,an elixir, a suspension, a syrup, a wafer, and the like, and in the caseof the formulation for injection, the pharmaceutical composition may beprepared in a form of a unit-dose or multi-dose ampoule or vial. Inaddition, the anti-cancer composition may contain a surfactant fortypically facilitating movement through a membrane. The surfactant asdescribed above may be derived from steroids, or may be a cationic lipidsuch as N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride(DOTMA), or the like, or various compounds such as cholesterolhemisuccinate, phosphatidyl glycerol, and the like.

In another aspect, the present invention provides a method of treatingcancer and suppressing growth of cancer by administering the compositioncontaining the anti-glypican 3 antibody according to the presentinvention to an individual. A pharmaceutically effective dose of thecomposition containing the anti-glypican 3 antibody according to thepresent invention may be administered in order to treat cancer cells ormetastasis thereof, or suppress growth of cancer. The pharmaceuticallyeffective dose may be changed by various factors such as the kind ofcancer, an age, a weight, characteristics and degree of symptoms of apatient, the kind of current treatment method, a treatment frequency, anadministration form and route, and the like, and may be easilydetermined by a specialist in the corresponding field. The compositionaccording to the present invention may be administered together with thepharmacological or physiological ingredient, or sequentiallyadministered. In addition, the composition according to the presentinvention and an additional therapeutic agent according to the relatedart may be combined with each other, and sequentially or simultaneouslyadministered with each other. Administration as described above may besingle dose administration or multi-dose administration. It is importantto administer the composition at a minimum dose while obtaining amaximum effect without side effects in consideration of all the factors,which may be easily determined by those skilled in the art.

As used herein, the term “individual” means a mammal, preferably, aperson in a state or with a disease, which may be alleviated,suppressed, or treated by administrating the humanized antibody orhaving the risk of the disease.

As used herein, the term “administration” means that a predeterminedmaterial is introduced to the individual by an appropriate method, andthe composition containing the humanized antibody according to thepresent invention may be administered through any general route as longas a drug may arrive at a target tissue through the route. Examples ofthe administration include intraperitoneal administration, intravenousadministration, intramuscular administration, subcutaneousadministration, intradermal administration, oral administration, topicaladministration, intranasal administration, intrapulmonaryadministration, and rectal administration, but are not limited thereto.However, since proteins are digested at the time of oral administration,it is preferable that the composition for oral administration is coatedwith an active drug or formulated so as to be protected from degradationin the stomach. In addition, the pharmaceutical composition may beadministered by an optional device capable of moving an active materialto target cells.

Hereinafter, the present invention will be described in detail throughthe Examples. However, these Examples are only to illustrate the presentinvention, and those skilled in the art will appreciate that theseExamples are not to be construed as limiting a scope of the presentinvention.

Example 1: Preparation of Anti-Glypican 3 Antibody

1-1: Selection of Anti-Human GPC3 scFv Antibody Using Phage Display

An antibody selection process was performed using a phage displaytechnology of inserting a DNA sequence to be expressed into a genome ofa bacteriophage parasitic on E. coli using a genetic recombinanttechnology and expressing the inserted protein on a surface of the phagein a form in which the protein is fused with one of coating proteins ofthe phage. At the time of primary panning, 1 ml of library stock (10¹³or more) was reacted in a GPC3-coated solid phase polystyrene tube(Nunc, 444202) at 37° C. for 2 hours. At the same time, 10 ul ofXLI-Blue electroporation-competent cell (Stratagene) was inoculated intoSB (10 ml)/tetracycline (100) and cultured so that OD600 was 0.8 to 1.The reactant at 37° C. for 2 hours was washed with 0.05% Tween 20/PBS (5ml) four times, and from the secondary panning, as the number of panningwas increased, the number of washing with 0.05% Tween 20/PBS (5 ml) wasincreased. Thereafter, the resultant was cultured in 1% BSA/0.1M Glycine(pH 2.0) at room temperature for 10 minutes, and phagemid was purified.The purified phagemid was transferred into a 50 ml tube and neutralizedwith 2M tris (70 ul). 9 ml of XLI-Blue Electroporation-Competent Cell(Stratagene) was treated, and 1 ml of XLI-Blue Electroporation-CompetentCell (Stratagene) was treated to the washed tube. After infection atroom temperature for 30 minutes, SB (10 ml), tetracycline (20 μl), andcarbenicillin (10 μl) were added thereto, and the infected cells weresuspension-cultured at 37° C. and 220 rpm for 1 hour. Then, the culturedcells were treated with 1 ml of VCS M13 helper phage (1011 pfu),suspension-cultured at 37° C. and 220 rpm for 1 hour, treated with SB(80 ml), kanamycin (100 μl), and carbenicillin (100 μl), and cultured at37° C. and 220 rpm for 12 hours or more. The cells cultured for 12 hoursor more were centrifuged at 4° C. and 3500 rpm for 10 minutes, asupernatant was transferred to a new tube and 20% PEG/15% NaCl (20 ml)was added thereto and well-mixed, followed by reaction on ice for 30minutes. Next, a supernatant was removed by centrifugation at 4° C. and8000 rpm for 30 minutes, and the remaining pellet was collected andre-suspended in 1% BSA/PBS (2 ml), followed by centrifugation at 4° C.and 15000 rpm for 10 minutes. At this time, the collected pellet wasremoved and 1 ml of a total of 2 ml of supernatant was stored at −20°C., and 1 ml of the supernatant was used in the next panning.

1-2: Securing Individual Clone Using ELISA Method

A single colony from final amplification group of a phage displaysynthetic scFV library was collected, cultured in SB/carbenicillin (1.5ml) at 37° C. and 220 rpm until OD 600 was 0.8 to 1.0 or so, and thencultured in 1 mM IPTG at 30° C. and 200 rpm for 12 hours or more. Afterthe reactant was centrifuged at 5500 rpm for 5 minutes, only each of thesupernatants was added to an ELISA plated with a GPC3 antigen, reactedat room temperature for 2 hours, and washed with PBST (1×PBS, 0.05%Tween 20) four times. Then, after a HRP/Anti-hFab-HRP conjugate dilutedwith 1% BSA/1×PBS at a ratio of 1/5000 was added thereto, reacted atroom temperature for 1 hour, and washed with PBST (1×PBS, 0.05% Tween20) four times again, a TMB solution was added thereto, and a reactionwas carried out for 5 to 10 minutes. After adding the TMB stop solutionthereto, an OD value was measured at a wavelength of 450 nm using aTECAN sunrise, a clone having a high OD value was secured as anindividual clone.

As a result, 61 kinds of clones specifically binding to human GPC3 wereselected, and amino acid sequences thereof were secured. The selectedclones were named as clone GX090, clone GX092, clone GX099, clone GX102,clone GX107, clone GX114, clone GX116, clone GX118, clone GX119, cloneGX122, clone GX184, clone GX186, clone GX189, clone GX196, clone GX197,clone GX201, clone GX205, clone GX206, clone GX207, clone GX209, cloneGX213, clone GX214, clone GX216, clone GX217, clone GX219, clone GX221,clone GX222, clone GX224, clone GX225, clone GX226, clone GX229, cloneGX233, clone GX234, clone GX235, clone GX242, clone GX245, clone GX247,clone GX248, clone GX253, clone GX259, clone GX263, clone GX264, cloneGX265, clone GX268, clone GX270, clone GS001, clone GS002, clone GS003,clone GS004, clone GS005, clone GS006, clone GS007, clone GS008, cloneGS009, clone GS010, clone GS011, clone GS012, clone GS013, clone GS014,clone GS015, clone GS016, clone GS017, clone GS018, clone GN328, cloneGN337, and clone GN414, respectively. A variable region sequence of eachof the clones was confirmed as illustrated in Table 1, and a CDR aminoacid sequence in the variable region of each of the clones was confirmedas illustrated in Table 2 based on Kabat numbering.

TABLE 1 SEQ Variable ID Clone Region Amino Acid Sequencce NO: GX090Heavy QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQG 1 ChainLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARGSSFSGFDPWGQGTLVTVSS LightQSVLTQPPSLSAAPGQKVTISCSGSSSNIGKNHVSWYQQFPGTA 2 ChainPKFLIYDNHRRPSGIPDRFSGSKSGTSATLDITGLQTGDEATYY CGTWDSSLSAVVFGGGTKLTVLGGX092 Heavy QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQG 3 ChainLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARADWGFFDYWGQGTLVTVSS LightQSVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVTWYQQLPGTA 4 ChainPKLLIIYANNQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYY CAAWDDSINGWVFGGGTKLTVLGGX099 Heavy EVQLVESGAEVKKPGASVKVSCKASGYTFTSYYMHMVROAFGQG 5 ChainLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEETAVYYCARYSSSPAKIDYWGQGTLVTVSS LightQSVLTQPPSVSAAPGEKVIISCSGSSSNIGKYYVSWYQQLPGTA 6 ChainPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGISGLQTGDEADYY CGTWDSSLNLVFGTGTKVTVLGGX102 Heavy QMQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQG 7 ChainLEWMGWINPNSGGTNYAQKFOGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARRGLRGDFDYWGQGTLVTVSS LightGQSVLTQPPSASETPGQKVTISCSGSSSNIGTNHVFWYQQLPGT 8 ChainAPKLLIYRNNLRPSGVPDRFSGSKSGTSASLAISGLRSEDEADY YCAAWDDSLSWVFGGGTKLTVLGGX107 Heavy QMQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQG 9 ChainLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSHGDYPEDYWGQGTLVTVSS LightYELTQPPSVSVSPGQTARIACSGDALPKHYAYWYQQKSGQAPVL 10 ChainVVYEDKKRPAGIPERFSGSSSGTVATLTISGAQVEDEAHYYCYS TDTSGNHRVFGGGTKLTVLG GX114Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKG 11 ChainLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARVVDSSDYWGQGTLVTVSS LightAIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAP 12 ChainKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYC QQYDNLPLTFCGGTKVEIKR GX116Heavy QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQG 13 ChainLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARVMVRGVSTFGYWGQGTLVTVSS LightQSVLTQPPSVSAAPGQKVSISCSGSSSNIGKNFLSWYQQLPGTA 14 ChainPHLLIYRNNRRPSGIPDRFSGSKSGTSATLTITGLQTGDEGDYY CGAWDSRLSGVVFGGGTKLTVLGGX118 Heavy QLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPG 15 ChainKGLEWIGSIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARLRSFADNYVWGSYASYYFDYWGRGTLVTVSS LightQSVVTQPPSVSAAPGQRVTISCSGSSSNIGNNFVSWHQQLPGTA 16 ChainPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYY CGTWDNSLGTGVFGGGTKLTVLGGX119 Heavy EVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKG 17 ChainLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRSWNYYGMDVWGQGTTVTVSS LightAIRMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAP 18 ChainKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQSYSTPVTFGGGTKLEIKR GX122Heavy EVQLVESGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQG 19 ChainLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDGGSSPDIWGQGTMVTVSS LightSYELTQPPSVSVSPGQTARITCSGDALPKKYAYWYQQKSGQAPV 20 ChainLVIYEDKKRPSEIPERFSGSSSGTMATLTITGAQVDDEADYYCY SIDRSGSRGVFGGGTKLTVLG GX184Heavy QLQLQESGPGLVEPSETLSLTCAVSGGSISSSNNYWGWIRQPPG 21 ChainKGLEWIGSIFYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARLPTGTPGFYFDYWGQGTLVTVSS LightSYELTQPPSVSKGLRQTATLTCTGNSNNVGNQGAAWLQQHQGHP 22 ChainPKLLSYRNNNRPSGISERFSASRSGNTASLTITGLQPEDEADYY CSAWDSSLSAWVFGGGTKLTVLGGX186 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKG 23 ChainLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRA EDTAVYYCARLQGYWGRGTLVTVSSLight DIQMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQ 24 ChainKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAED VAVYYCQQYYSTPPTFGGGTKLEIKRGX189 Heavy QLQLQESGPGLVKPSETLSLTCAVSGGSISSSNNYWGWIRQPPG 25 ChainKGLEWIGSIFYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARLPTGTPGFYFDYWGQGTLVTVSS LightSYVLTQPPSVSKGLRQTATLTCTGNSSNVGNQGAAWLQQHQGHP 26 ChainPKLLSYRNNNRPSGISERFSASKSGNTASLTITGLRPEDEADYY CSAWDSSLSAWVFGGGTKLTVLGGX196 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKG 27 ChainLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRD EDTAVYYCARVTGDYWGRGTLVTVSSLight EIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQ 28 ChainKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAED VAVYYCOQYYSTPLIFGGGTKVEIKRGX197 Heavy QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQG 29 ChainLEWMGWISAYNGNTNYAQKLQGRVIMTTDTSTSTAYMELRSLRSDDTAVYYCASQGSGWLDYWGQGTLVTVSS LightNFMLTQPHSVSESPGKTVTISCTGSGGNIASNYVQWFQQRPGSA 30 ChainPTTVIYDDVQRPSGVPNRFSGSIDSSSNSASLSISGLKTEDEAD YYCQSYDRTYRGVFGGGTKLTVLGGX201 Heavy QVQLVESGGGLVNPGGSLRLSCAASGFTFSNYGMNWVRQAPGKG 31 ChainLEWVSSISTRSGYIFYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDRPNRSGMDVWGQGTTVTVSS LightQSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGK 32 ChainAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEGDY YCSSYTSSDTLVFGSGTKVTVLGGX205 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKG 33 ChainLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRA EDTAVYYCARVLLDYWGQGTLVTVSSLight AIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKFGKAP 34 ChainKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYC QQYDNLLLTFGGGTKLEIKR GX206Heavy QLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPG 35 ChainKGLEWIGSIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARLGDYGGNGYYFDYWGQGTLVTVSS LightQSVLTQPPSVSAASGQKVTVSCSGSSSNIGKNGVSWHQQLPGTA 36 ChainPKLLIYENNRRPSEIPDRFSGSKSGTSATLAITGLQTGDEADYY CGTWDSSLNAGVFGTGTKVTVLGGX207 Heavy EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQG 37 ChainLEWMGWISAYNGNTNYAQKLQGRVTMITDTSTSTAYMELRSLRSDDTAVYYCAREGIAAAGYYYGMDVWGQGTTVTVSS LightQSALTQPPSVSGSPGQSVTISCTGTSSDVGGYNRVSWYQQPPGT 38 ChainAPKLMIYEVSNRPSGVPDRFSGSKSGNTASLTISGLQAEDEADY YCSSYTSSTTYVFGTGTKVTVLGGX209 Heavy QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQG 39 ChainLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGYYYGMDVWGQGTTVTVSS LightQSVLTQPPSASVSPGQTASITCSGDKLGDKYVFWYRQKPGQSPV 40 ChainLVIYQDNKRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCQ TWDSSTEVFGTGTKVTVLG GX213Heavy QLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPG 41 ChainKGLEWIGSIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARLFWQQLTFDYWGQGTLVTVSS LightQSVVTQPPSVSAAPGQRVSISCSGSSSNIGKNHVIWHQQFPGTA 42 ChainPKLLISENNKRPSGIPDRFSASKSGTSATLDITGLQTGDEADYY CGTWDNSLSAGVFGGGTKLTVLGGX214 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKG 43 ChainLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRV DDTAVYYCATLYDFWSQGTLVTVSSLight NIQMTQSPSSLSASVGDRVTITCQASQDITNFLNWHQQKPGKAP 44 ChainELLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDVGTYYC QQYDNLPLTFGGGTKVEIKR GX216Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKG 45 ChainLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARVVDSSDYWGQGTLVTVSS LightEIVLTQSPATLSLSPGESATLSCRASQSVSSYLAWYQQKPGQAP 46 ChainRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYC QQRSNWPPVALTFGGGTKLEIKRGX217 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKG 47 ChainLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARVSGDPKDWGQGTLVTVSS LightDIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAP 48 ChainRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYC QQYNNWPRELFGPGTKVDIKR GX219Heavy QMQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQG 49 ChainLEWMGRINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARVARYCSSTSCRTGGMDVWGQGTTVTVSS LightQSVLTQPPSASGTPGQRVTISCSGRSSNIGSRYVYWYQQLTGTA 50 ChainPKLLIFRNDQRPSGVPDRFSASKSGTSASLAISGLRSEDEADYF CATWDDSLSAWVFGGGTKLTVLGGX221 Heavy QLQLQESGPGLVKPSETLSLTCAVSGGSISSSNNYWGWIRQPPG 51 ChainKGLEWIGSIFYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARLPTGTPGFYFDYWGQGTLVTVSS LightSYVLTQPPSVSKDLRQTATLTCTGNSSNVGNQGAAWVQQHQGHP 52 ChainPKLLSYRNNKRPSGISERLSASRSGNTASLTITGLQPEDEADYY CSAWDSSLSAWVFGGGTKLTVLGGX222 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKG 53 ChainLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRA EDTAVYYCARVTLDYWGRGTLVTVSSLight AIRMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAP 54 ChainKLLIYDASNLETGVPSRFSGSGSETDFTFTISSLQPEDIATYYC QQYDSLPTTFGPGTKVDIKR GX224Heavy QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQG 55 ChainLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSRPRSSSFDYWGRGTLVTVSS LightNFMLTQPHSVSESPGKTVTISCTVSSGSIAKNYVHWYQRRPGSA 56 ChainPTPLIYEDNRRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEAD YYCQSYDDSGDRFVFGTGTKVTVLGGX225 Heavy QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQG 57 ChainLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARESGPSGGMDVWGQGTTVTVSS LightSYELTQPPSVSVSPGQTARITCSGDALPKQYAYWYQQKPGQAPV 58 ChainLVIYKDSERPSGIPERFSGSSSGTTVTLTISGVQAEDEADYYCS SYTSSSTRVVFGGGTKLTVLG GX226Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKG 59 ChainLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNTLYLQMNSLRA EDTAVYYCARVTLDYWGRGTLVTVSSLight DIVMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAP 60 ChainKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYC QQYDNLPSITFGQGTRLEIKR GX229Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKG 61 ChainLEWVSYISSSGSTIYYADSVKGRFTISRDNSKNTLYLQMNSLRA EDTAVYYCARVLLDYWGQGTLVTVSSLight DIVMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAP 62 ChainKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYC QQYDNLWLTFGGGTKLEIKR GX233Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKG 63 ChainLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARVVDSSDYWGQGTLVTVSS LightAIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAP 64 ChainKLLIYDASNLETGVPSRFSGSGSGTDFTLRISRVEAEDVGIYYC MEVRYWPYNFGQGTKLEIKR GX234Heavy QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQG 65 ChainLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARVGLGDFWSGDYYYYYYGMDVWGQGTTVTVSS LightSSELTQDPAVSVALGQTVRITCQGDSLRSSYASWYHQKPGQAPV 66 ChainLVMYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCH SRDSTGSHPNWVFGGGTKLTVLGGX235 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKG 67 ChainLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARVSGDPKDWGQGTLVTVSS LightAIQMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQ 68 ChainKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLXAED VAVYYCQQYYSTPLTFGGGTKLEIKRGX242 Heavy QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKG 69 ChainLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARVVAAREDYWGQGTLVTVSS LightEIVLTQSPATLSLSPGERATLSCRATQSVGSYLAWYQQKPGQAP 70 ChainRLLIYDAFNRATGIPDRFSGSGSGTDFTLTISSLEPEDFAVYYC QQRSNWPLTFGGGTKVEIKR GX245Heavy QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKG 71 ChainLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARVSDDSPYWGQGTLVTVSS LightVIWMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAP 72 ChainKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYC QQYDNLLALTFGGGTKLEIKR GX247Heavy QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQG 73 ChainLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARTRKGSGYSYGYGMDVWGQGTTVTVSS LightSYELTQPPSVSVSPGQTASITCSGDKLGNKYVSWYQQKPGQSPV 74 ChainLVIYQDNKRPSGIPERFSGSNSGNTATLTISGTQAMDEAGYYCQ TWDSSVVFGGGTKLTVLG GX248Heavy QMQLVQSGAEVKKPGASVKVSCKASGGTFSSYAISWVRQAPGQG 75 ChainLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARVRGTGPRGGTFDYWGQGTLVTVSS LightQSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTA 76 ChainPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYY CGTWDSSLSAWVFGGGTKLTVLGGX253 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKG 77 ChainLEWVSYISSSGTTIYYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTAVYYCARVRDSSGFWGRGTLVTVSS LightQSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTA 78 ChainPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYY CGTWDSSLSAWVVGGGTKLTVLGGX259 Heavy QVQLVQSGAEVKKPGESLRISCKASGYKFTNYWIAWVRQMPGKG 79 ChainLEWMGIIYPGDSDTRYSPSLQGQVTISADKSITTAYLQWSSLRASDTAMYYCATIGLREGRWGQGTLVTVSS LightAIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQYPGKAP 80 ChainKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYC QQYDNTLPFGQGTKLEIKR GX263Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKG 81 ChainLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARVTPYYYYGMDVWGQGTTVTVSS LightQSVVTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTA 82 ChainPKLLIYDSNKRPPGIPDRFSGSKSGTSATLGITGLQTGDEADYY CGAWDSSLSAVVFGGGTKLTVLGGX264 Heavy QLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPG 83 ChainKGLEWIGSIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARLRWYSYGYFDVRGGYYFDYWGRGTLVTVSS LightQSVLTQPPSVSAASGQKVTVSCSGSSSNIGKNGVSWHQQLPGTA 84 ChainPKLLIYENNRRPSEIPDRFSGSKSGTSATLAITGLQTGDEADYY CGTWDSSLNAGVFGTGTKVTVLGGX265 Heavy QVQLVQSGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKG 85 ChainLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRA EDTAVYYCARVHLDYWGQGTLVTVSSLight DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQ 86 ChainKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAED VAVYYCQQYYSTPPTFGQGTKVEIKRGX268 Heavy EVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKG 87 ChainLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRSWNYYGMDVWGQGTTVTVSS LightNIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAP 88 ChainKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQSYSTPTFGQGTRLEIKR GX270Heavy QVQLVQSGAEVKKPGASVKVSCKTSGYTFTSSYMHWVRQAPGQG 89 ChainLEWMGIINPNSRGTNYAQKFQGRVTMTSDTSISTAYMELSSLISDDTAVYYCASSSGDYPDYWGQGTLVTVSS LightSYELTQPPSVSVSPGQTARIACSGDALPKHYAYWYQQKSGQAPV 90 ChainLVVYEDKKRPAGIPERFSGSSSGTVATLTISGAQVEDEAHYYCY STDTSGNHRVFGGGTKLTVLG GS001Heavy EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYSMSWVRQAPGKG 91 ChainLEWVSGISPSGGNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARALLGCKSAYCYYAMDVWGQGTLVTVSS LightQSVLTQPPSASGTPGQRVTISCTGSSSNIGSNAVNWYQQLPGTA 92 ChainPKLLIYADSKRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYY CGSWDSSLSGYVFGGGTKLTVLGGS002 Heavy EVQLLESGGGLVQPGGSLRLSCAASGFTFSGYSMSWVRQAPGKG 93 ChainLEWVSSISPGGSSTYYADSVKGRFTISRDNSKNTLYLQMSSLRAEDTAVYYCARYRVWHMTTGDYYSNAMDVWGQGTLVTVSS LightQSVLTQPPSASGTPGQRVTISCTGSSSNIGSNAVSWYQQLPGTA 94 ChainPKLLIYDDNKRPSGVLDRFSGSKSGTSASLAISGLRSEDEADYY CGTWDDSLSGYVFGGGTKLTVLGGS003 Heavy EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYSMSWVRQAPGKG 95 ChainLEWVSVISPGSGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYRVMRSPHNPYYSYGMDVWGQGTLVTVSS LightQSVLTQPPSASGTPGQRVTISCSGSSSNIGSNDVNWYQQLPGTA 96 ChainPKLLIYSNSQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYY CGSWDASLSGYVFGGGTKLTVLGGS004 Heavy EVQLLESGGGLVQPGGSLRLSCAVSGFTFSNYSMSWVRQAPGKG 97 ChainLEWVSAISPGSSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYRVWAHSASSSYSNAMDVWGQGTLVTVSS LightQSVLTQPPSASGTPGQRVTISCSGSSSNIGNNDVSWYQQLPGTA 98 ChainPKLLIYSDSQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYY CGSWDDSLNGYVFGGGTKLTVLGGS005 Heavy EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKG 99 ChainLEWVSVISPDSSSKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARAAVRRYGPSPYYYYAMDVWGQGTLVTVSS LightQSVLTQPPSASGTPGQRVTISCTGSSSNIGNNYVTWYQQLPGTA 100 ChainPKLLIYADSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYY CATWDYSLSGYVFGGGTKLTVLGGS006 Heavy EVQLLESGGGLVQPGGSLRLSCAASGFTFSGYSMSWVRQAPGKG 101 ChainLEWVSAISPGGGSKYYADSVRGRFTVSRDNSKNTLYLQMNSLRAEDTAVYYCARYRVQKSAKNVYSSNGMDVWGQGTLVTVSS LightQSVLTQPPPASGTPGQRVTISCSGSSSNIGNNAVNWYQQLPGTA 102 ChainPKLLIYYDSNRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYY CGAWDSSLSAYVFGGGTKLTVLGGS007 Heavy EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYSMSWVRQAPGKG 103 ChainLEWVSVISHDGRSKYYADSVKGRFAISRDNSKNTLYLQMNSLRAEDTAVYYCAKFRVWKRTNAHSYANAMDVWGQGTLVTVSS LightQSVLTQPPSASGTPGQRATISCTGSSSNIGSNSVSWYQQLPGTA 104 ChainPKLLIYANSNRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYY CGTWDASLSAYVFGGGTKLTVLGGS008 Heavy EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKG 105 ChainLEWVSVISPDNGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARARVHCRRDQCYSYGMDVWGQGTLVTVSS LightQSVLTQPPSASGTPGQRVTISCTGSSSNIGSNNVTWYQQLPGTA 106 ChainPKLLIYANSQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYY CGAWDSSLNGYVFGGGTKLTVLGGS009 Heavy EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYSMSWVRLAPGKG 107 ChainLEWVSVISPSGGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEHTAVYYCARYRVKQMLNQRSYSNAMDVWGQGTLVTVSS LightQSVLTQPPSASGTPGQRVTISCTGSSSNIGNNYVSWYQQLPGTA 108 ChainPKLLIYDDSQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYY CGSWDASLSGYVFGGGTKLTVLGGS010 Heavy EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYSMSWVRQAPGKG 109 ChainLEWVSVISPSSGSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYRVRQLKHTRSYADAMDVWGQGTLVTVSS LightQSVLTQPPSASGAPGQRVTISCSGSSSNIGSNAVSWYQQLPGTA 110 ChainPKLLIYANSHRPSGVPDRFSGSKSGTSASLAISGLRSEDETDYY CASWDSSLSGYVFGGGTKLTVLGGS011 Heavy EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKG 111 ChainLEWVSGISPGGSSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYAVYFLRSHGSYDYGMDVWGQGTLVTVSS LightQSVLTQPPSASGTPGQRVTISCTGSSSNIGSNDVTWYQQLPGTA 112 ChainPKLLIYSNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYY CGAWDYSLNAYVFGGGTKLTVLGGS012 Heavy EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYSMSWVRQAPGKG 113 ChainLEWVSGISPGSGSKYYADSVRGRFTVSRDNSKNTLYLQMNSLRAEDTAVYYCARRARRFDYWGQGTLVTVSS LightQSVLTQPPSASGTPGQRVTISCTGSSSNIGNNAVTWYQQLPGTA 114 ChainPKLLIYSNSQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYY CGSWDSSLNGYVFGGGTKLTVLGGS013 Heavy EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKG 115 ChainLEWVSSISSGGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARKKSQFDYWGQGTLVTVSS LightQSVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQLPGTA 116 ChainPKLLIYANSQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYY CDTWDYSLSGYVFGGGTKLTVLGGS014 Heavy EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKG 117 ChainLEWVSVISPNSGSNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARPRILRRRVDHSYSYAMDVWGQGTLVTVSS LightQSVLTQPPSASGTPGQRVTISCTGSSSNIGNNYVSWYQQLPGTA 118 ChainPKLLIYSNSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYY CGTWDYSLSGYVLGGGTKLTVLGGS015 Heavy EVOLLESGGGLAQPGGSLRLSCAASGFTFSDYSMSWVRQAPGKG 119 ChainLEWVSAISPDGGSKYYADSVKGRFTISRDNSKNTLYLQWNSLRAEDTAVYYCARFRVIKLRAGWYSANGMDVWGQGTLVTVSS LightQSVLTQPPSASGTPGQRVTISCSGSSSNIGNNDVSWYQQLPGTA 120 ChainPKLLIYYNSKRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYY CGAWDDSLNGYVFGGGTKLTVLGGS016 Heavy EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKG 121 ChainLEWVSGIYSGNGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARALSSCPRGPCYYDDGMDVWGQGTLVTVSS LightQSVLTQPPSASGTPGQRVTISCSGSSSNIGSNDVIWYQQLPGTA 122 ChainPKLLIYDNSKRPSGVPDRFSGSKSGTSASLXISGLRSGDEADYY CGTWDASLSAYVFGGGTKLTVLGGS017 Heavy EVQLLESGGGLVQPGGSLRLSCAASGFTFSGYAMSWVRQAPGKG 123 ChainLEWVSGISPGDGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVARMCQGWRCSYADGMDVWGQGTLVTVSS LightQSVLTQPPSASGTPGQRVTISCTGSSSNIGNNSVYWYQQLPGTA 124 ChainPKLLIYSDSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYY CGTWDSSLSGYVFGGGTKLTVLGGS018 Heavy EVQLLESGGGLVQPGGSLRLSCAASGLTFSNYAMSWVRQAPGKG 125 ChainLEWVSVISPGSGSKYYADSVKGRFTVSRDNSKNTLYLQMNSLRAEDTAVYYCARHRVIKINRQTYYDYGMDVWGQGTLVTVSS LightQSVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVSWYQQLPGTA 126 ChainPKLLIYSDNNRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYY CGAWDDSLSAYVFGGGTKLTVLGGN328 Heavy EVQLVESGGGLVQPGGSLRVSCAASGFTFSTAWMDWVRQAPGKG 127 ChainLEWVANINPDGSEKHYVDSVKGRFTVSRDNAKNSVYLHMNTLRA EDTAVYYCSKALDYWGQGTTVIVSSLight DIQMTQSPSTLSASVGDRVTITCRASQNINTWLAWYQQKPGKAP 128 ChainEVLIYEASSLESGVPPRFSGSGSGTEFTLTISSLQPDDSATYYC QQYNTYSPTFGQGTKLEIKR GN337Heavy EVQLVESGPGLVKPSQTLSLTCTVSGGSVSSGGYHWNWIRQHPG 129 ChainKGLEWIGYIFNSGNTDYNPSLRSRLTISQDTSKNQFSLKLSSVTAADTAVYYCARHRSALLRWFDYWGQGTMVTVSSASTKGPSVFPL APSSKSTSG LightDVVLTQSPSSLSAFVGDRVTITCQASQDIRKYLNWFQQKPGKAP 130 ChainKLLIYDASTLETGVSSRFSGSGSGTHFNLVISSLEPEDSATYYC QQHDFVPRTFGQGTKLEIKR GN414Heavy QVQLVQSGAEVKKPGASVKVSCKASGYTFSTYGITWVRQAPGQG 131 ChainLEWMGRITPNNGVTDYAQKFQGRVTMTRDTSISTAYMELSSLRSDDSAVYYCAREIGSSSWKLLDPWGQGTMVTVSS LightQAVLTQPSSVSVSPGQTASITCTGNSNNVGYEGAAWVQQYQGHP 132 ChainPKLLSDRNHNRPSGISERFSASRSGNTASLTITGLQSEDEADYY CSAWDSSLSEWVSGGGTKLTVLG

TABLE 2 Variable Clone Region CDR1 CDR2 CDR3 GX090 Heavy SYGISWISAYNGNTNYAQKLQG GSSFSGFDP Chain (SEQ ID NO: (SEQ ID NO: 134) (SEQ IDNO: 135) 133) Light SGSSSNIGKNHVS DNHRRPS GTWDSSLSA Chain (SEQ ID NO:(SEQ ID NO: 137) (SEQ ID NO: 138) 136) GX092 Heavy SYGISWISAYNGNTNYAQKLQG ADWGFFDY Chain (SEQ ID NO: (SEQ ID NO: 134) (SEQ IDNO: 139) 133) Light SGSSSNIGSNTVT ANNQRPS AAWDDSLNG Chain (SEQ ID NO:(SEQ ID NO: 141) (SEQ ID NO: 142) 140) GX099 Heavy SYYMHIINPSGGSTSYAQKFQG YSSSPAKIDY Chain (SEQ ID NO: (SEQ ID NO: 144) (SEQ IDNO: 145) 143) Light GSSSNIGKYYVS DNNKRPS GTWDSSLNA Chain (SEQ ID NO:(SEQ ID NO: 147) (SEQ ID NO: 148) 146) GX102 Heavy GYYMHWINPNSGGTNYAQKFQG RGLRGDFDY Chain (SEQ ID NO: (SEQ ID NO: 150) (SEQ IDNO: 151) 149) Light SGSSSNIGTNHVF RNNIRPS AAWDDSLS Chain (SEQ ID NO:(SEQ ID NO: 153) (SEQ ID NO: 154) 152) GX107 Heavy SYYMHIINPSGGSTSYAQKFQG SHGDYPEDY Chain (SEQ ID NO: (SEQ ID NO: 144) (SEQ IDNO: 155) 143) Light SGDALPKHYAY EDKKRPA YSTDTSGNH Chain (SEQ ID NO: (SEQID NO: 157) (SEQ ID NO: 158) 156) GX114 Heavy DYYMS YISSSGSTIYYADSVKGVVDSSDY Chain (SEQ ID NO: (SEQ ID NO: 160) (SEQ ID NO: 161) 159) LightQASQDISNYLN DASNLET QQYDNLP Chain (SEQ ID NO: (SEQ ID NO: 163) (SEQ IDNO: 164) 162) GX116 Heavy SYYMH IINPSGGSTSYAQKFQG VMVRGVSTFGY Chain (SEQID NO: (SEQ ID NO: 144) (SEQ ID NO: 165) 143) Light SGSSSNIGKNFLSRNNRRPS GAWDSRLSG Chain (SEQ ID NO: (SEQ ID NO: 167) (SEQ ID NO: 168)166) GX118 Heavy SSSYYWG SIYYSGSTYYNPSLES LRSFADNYV Chain (SEQ ID NO:(SEQ ID NO: 170) (SEQ ID NO: 171) 169) Light SGSSSNIGNNFVS DNNERPSGTWDNSLGT Chain (SEQ ID (SEQ ID NO: 147) (SEQ ID NO: 173) NO: 172) GX119Heavy SYGMH VIWYDGSNKYYADSVKG DRSWNYYGMDV Chain (SEQ ID NO: (SEQ ID NO:175) (SEQ ID NO: 176) 174) Light RASQSISSYLN AASSLQS QQSYSTP Chain (SEQID NO: (SEQ ID NO: 178) (SEQ ID NO: 179) 177) GX122 Heavy SYYMHIINPSGGSTSYAQKFQG DGGSSPDI Chain (SEQ ID NO: (SEQ ID NO: 144) (SEQ IDNO: 180) 143) Light SGDALPKKYAY EDKKRPSEI YSIDRSGSR Chain (SEQ ID NO:(SEQ ID NO: 182) (SEQ ID NO: 183) 181) GX184 Heavy SSNNYWGSIFYSGSTYYNPSLKS LPTGTPGFYFDY Chain (SEQ ID NO: (SEQ ID NO: 185) (SEQ IDNO: 186) 184) Light TGNSNNVGNQGAA RNNNEPS SAWDSSLSA Chain (SEQ ID NO:(SEQ ID NO: 392) (SEQ ID NO: 188) 187) GX186 Heavy DYYMSYISSSGSTIYYADSVKG LQGY Chain (SEQ ID NO: (SEQ ID NO: 160) (SEQ ID NO:189) 159) Light KSSQSVLYSSNNKN WASTRES QQYYSTP Chain YLA (SEQ ID NO:(SEQ ID NO: 191) (SEQ ID NO: 192) 190) GX189 Heavy SSNNYWGSIFYSGSTYYNPSLKS LPTGTPGFYFDY Chain (SEQ ID NO: (SEQ ID NO: 185) (SEQ IDNO: 186) 184) Light TGNSSNVGNQGAA RNNNRPS SAWDSSLSA Chain (SEQ ID NO:(SEQ ID NO: 392) (SEQ ID NO: 188) 193) GX196 Heavy DYYMSYISSSGSTIYYADSVKG VTGDY Chain (SEQ ID NO: (SEQ ID NO: 160) (SEQ ID NO:194) 159) Light KSSQSVLYSSNNKN WASTRES QQYYSTP Chain YLA (SEQ ID NO:(SEQ ID NO: 191) (SEQ ID NO: 192) 190) GX197 Heavy SYGISWISAYNGNTNYAQKLQG QGSGWLDY Chain (SEQ ID NO: (SEQ ID NO: 134) (SEQ IDNO: 195) 133) Light TGSGGNIASNYVQ DDVQRPS QSYDRTYR Chain (SEQ ID NO:(SEQ ID NO: 197) (SEQ ID NO: 198) 196) GX201 Heavy NYGMNSISTRSGYIFYADSVKG DRPNRSGMDV Chain (SEQ ID NO: (SEQ ID NO: 200) (SEQ IDNO: 201) 199) Light TGTSSDVGGYNYVS DVSNRPS SSYTSSDT Chain (SEQ ID NO:(SEQ ID NO: 203) (SEQ ID NO: 204) 202) GX205 Heavy DYYMSYISSSGSTIYYADSVKG VLLDY Chain (SEQ ID NO: (SEQ ID NO: 160) (SEQ ID NO:205) 159) Light QASQDISNYLN DASNLET QQYDNLLA Chain (SEQ ID NO: (SEQ IDNO: 163) (SEQ ID NO: 206) 162) GX206 Heavy SSSYYWG SIYYSGSTYYNPSLKSLGDYGGNGYYFDY Chain (SEQ ID NO: (SEQ ID NO: 170) (SEQ ID NO: 207) 169)Light SGSSSNIGKNGVS ENNRRPS GTWDSSLNA Chain (SEQ ID NO: (SEQ ID NO: 209)(SEQ ID NO: 148) 208) GX207 Heavy SYGIS WISAYNGNTNYAQKLQG EGIAAAGYYYGMDVChain (SEQ ID NO: (SEQ ID NO: 134) (SEQ ID NO: 210) 133) LightTGTSSDVGGYNRVS EVSNRPS SSYTSSTTY Chain (SEQ ID NO: (SEQ ID NO: 212) (SEQID NO: 213) 211) GX209 Heavy SYYMH IINPSGGSTSYAQKFQG GYYYGMDV Chain (SEQID NO: (SEQ ID NO: 144) (SEQ ID NO: 214) 143) Light SGDKLGDKYVF QDNKRPSQTWDSST Chain (SEQ ID NO: (SEQ ID NO: 216) (SEQ ID NO: 217) 215) GX213Heavy SSSYYWG SIYYSGSTYYNPSLKS LFWQQLTFDY Chain (SEQ ID NO: (SEQ ID NO:170) (SEQ ID NO: 218) 169) Light SGSSSNIGKNHVI ENNKRPS GTWDNSLSA Chain(SEQ ID NO: (SEQ ID NO: 220) (SEQ ID NO: 221) 219) GX214 Heavy DYYMSYISSSGSTIYYADSVKG LYDF Chain (SEQ ID NO: (SEQ ID NO: 160) (SEQ ID NO:222) 159) Light QASQDITNFLN DASNLET QQYDNLP Chain (SEQ ID NO: (SEQ IDNO: 163) (SEQ ID NO: 164) 223) GX216 Heavy DYYMS YISSSGSTIYYADSVKGVVDSSDY Chain (SEQ ID NO: (SEQ ID NO: 160) (SEQ ID NO: 161) 159) LightRASQSVSSYLA DASNRAT QQRSNWPPVA Chain (SEQ ID NO: (SEQ ID NO: 225) (SEQID NO: 226) 224) GX217 Heavy DYYMS YISSSGSTIYYADSVKG VSGDPKD Chain (SEQID NO: (SEQ ID NO: 160) (SEQ ID NO: 227) 159) Light RASQSVSSNLA GASTRATQQYNNWPR Chain (SEQ ID NO: (SEQ ID NO: 229) (SEQ ID NO: 230) 228) GX219Heavy GYYMH RINPNSGGTNYAQKFQG VARYCSSTSCRTGGMDV Chain (SEQ ID NO: (SEQID NO: 231) (SEQ ID NO: 232) 149) Light SGRSSNIGSRYVY RNDQRPS ATWDDSLSAChain (SEQ ID NO: (SEQ ID NO: 234) (SEQ ID NO: 235) 233) GX221 HeavySSNNYWG SIFYSGSTYYNPSLKS LPTGTPGFYFDY Chain (SEQ ID NO: (SEQ ID NO: 185)(SEQ ID NO: 186) 184) Light TGNSSNVGNQGAA RNNKRPS SAWDSSLSA Chain (SEQID NO: (SEQ ID NO: 236) (SEQ ID NO: 188) 193) GX222 Heavy DYYMSYISSSGSTIYYADSVKG VTLDY Chain (SEQ ID NO: (SEQ ID NO: 160) (SEQ ID NO:237) 159) Light QASQDISNYLN DASNLET QQYDSLP Chain (SEQ ID NO: (SEQ IDNO: 163) (SEQ ID NO: 238) 162) GX224 Heavy SYYMH IINPSGGSTSYAQKFQGSRPRSSSFDY Chain (SEQ ID NO: (SEQ ID NO: 144) (SEQ ID NO: 239) 143)Light TVSSGSTAKNYVH EDNRRPS QSYDDSGDR Chain (SEQ ID NO: (SEQ ID NO: 241)(SEQ ID NO: 242) 240) GX225 Heavy SYYMH IINPSGGSTSYAQKFQG ESGPSGGMDVChain (SEQ ID NO: (SEQ ID NO: 144) (SEQ ID NO: 243) 143) LightSGDALPKQYAY KDSERPS SSYTSSSTR Chain (SEQ ID NO: (SEQ ID NO: 245) (SEQ IDNO: 246) 244) GX226 Heavy DYYMS YISSSGSTIYYADSVKG VTLDY Chain (SEQ IDNO: (SEQ ID NO: 160) (SEQ ID NO: 237) 159) Light QASQDISNYLN DASNLETQQYDNLP Chain (SEQ ID NO: (SEQ ID NO: 163) (SEQ ID NO: 164) 162) GX229Heavy DYYMS YISSSGSTIYYADSVKG VLLDY Chain (SEQ ID NO: (SEQ ID NO: 160)(SEQ ID NO: 247) 159) Light QASQDISNYLN DASNLET QQYDNLW Chain (SEQ IDNO: (SEQ ID NO: 163) (SEQ ID NO: 248) 162) GX233 Heavy DYYMSYISSSGSTIYYADSVKG VVDSSDY Chain (SEQ ID (SEQ ID NO: 160) (SEQ ID NO:161) NO: 159) Light QASQDISNYLN DASNLET MEVRYWP Chain (SEQ ID NO: (SEQID NO: 163) (SEQ ID NO: 249) (162) GX234 Heavy SYGIS WISAYNGNTNYAQKLQGVGLGDFWSGDYYYYYYGMDV Chain (SEQ ID NO: (SEQ ID NO: 134) (SEQ ID NO: 250)133) Light QGDSLRSSYAS GKNNRPS HSRDSTGSHPN Chain (SEQ ID NO: (SEQ ID NO:252) (SEQ ID NO: 253) 251) GX235 Heavy DYYMS YISSSGSTIYYADSVKG VSGDPKDChain (SEQ ID NO: (SEQ ID NO: 160) (SEQ ID NO: 227) 159) LightKSSQSVLYSSNNEN WASTRES QQYYSTP Chain YLA (SEQ ID NO: (SEQ ID NO: 191)(SEQ ID NO: 192) 190) GX242 Heavy DYYMS YISSSGSTIYYADSVKG VVAAREDY Chain(SEQ ID NO: (SEQ ID NO: 160) (SEQ ID NO: 254) 159) Light RATQSVGSYLADAFNRAT QQRSNWP Chain (SEQ ID NO: (SEQ ID NO: 256) (SEQ ID NO: 257) 255)GX245 Heavy DYYMS YISSSGSTIYYADSVKG VSDDSPY Chain (SEQ ID NO: (SEQ IDNO: 160) (SEQ ID NO: 258) 159) Light QASQDISNYLN DASNLET QQYDNLLA Chain(SEQ ID NO: (SEQ ID NO: 163) (SEQ ID NO: 206) 162) GX247 Heavy SYYMHIINPSGGSTSYAQKFQG TRKGSGYSYGYGMDV Chain (SEQ ID NO: (SEQ ID NO: 144)(SEQ ID NO: 259) 143) Light SGDKLGNKYVS QDNKRPS QTWDSS Chain (SEQ ID NO:(SEQ ID NO: 216) (SEQ ID NO: 261) 260) GX248 Heavy SYAISGIIPIFGTANYAQKFQG VRGTGPRGGTFDY Chain (SEQ ID NO: (SEQ ID NO: 263) (SEQID NO: 264) 262) Light SGSSSNIGNNYVS DNNKRPS GTWDSSLSA Chain (SEQ ID NO:(SEQ ID NO: 147) (SEQ ID NO: 138) 265) GX253 Heavy DYYMSYISSSGTTIYYADSVKG VRDSSGF Chain (SEQ ID NO: (SEQ ID NO: 266) (SEQ ID NO:267) 159) Light SGSSSNIGNNYVS DNNKRPS GTWDSSLSA Chain (SEQ ID NO: (SEQID NO: 147) (SEQ ID NO: 138) 265) GX259 Heavy NYWI IIYPGDSDTRYSPSLQGIGLREGR Chain (SEQ ID NO: (SEQ ID NO: 269) (SEQ ID NO: 270) 268) LightRASQGISNYLA DASNLET QQYDN Chain (SEQ ID NO: (SEQ ID NO: 163) (SEQ ID NO:272) 271) GX263 Heavy SYSMN SISSSSSYIYYADSVKG VTPYYYYGMDV Chain (SEQ IDNO: (SEQ ID NO: 274) (SEQ ID NO: 275) 273) Light SGSSSNIGNNYVS DSNKRPPGAWDSSLSA Chain (SEQ ID NO: (SEQ ID NO: 276) (SEQ ID NO: 277) 265) GX264Heavy SSSYYWG SIYYSGSTYYNPSLKS LRWYSYGYFDVRGGYYFDY Chain (SEQ ID NO:(SEQ ID NO: 170) (SEQ ID NO: 278) 169) Light SGSSSNIGKNGVS ENNRRPSGTWDSSLNA Chain (SEQ ID NO: (SEQ ID NO: 209) (SEQ ID NO: 148) 208) GX265Heavy DYYMS YISSSGSTIYYADSVKG VHLDY Chain (SEQ ID NO: (SEQ ID NO: 160)(SEQ ID NO: 279) 159) Light KSSQSVLYSSNNKN WASTRES QQYYSTP Chain YLA(SEQ ID NO: (SEQ ID NO: 191) (SEQ ID NO: 192) 190) GX268 Heavy SYGMHVIWYDGSNKYYADSVKG DRSWNYYGMDV Chain (SEQ ID NO: (SEQ ID NO: 175) (SEQ IDNO: 176) 174) Light RASQSISSYLN AASSLQS QQSYST Chain (SEQ ID NO: (SEQ IDNO: 178) (SEQ ID NO: 280) 177) GX270 Heavy SSYMH IINPNSRGTNYAQKFQGSSGDYPDY Chain (SEQ ID NO: (SEQ ID NO: 282) (SEQ ID NO: 283) 281) LightSGDALPKHYAY YEDKKRPA YSTDTSGNH Chain (SEQ ID NO: (SEQ ID NO: 284) (SEQID NO: 158) 156) GS001 Heavy SYSMS GISPSGGNKYYADSVKG ALLGCKSAYCYYAMDVChain (SEQ ID NO: (SEQ ID NO: 286) (SEQ ID NO: 287) 285) LightTGSSSNIGSNAVN ADSKRPS GSWDSSLSG Chain (SEQ ID NO: (SEQ ID NO: 289) (SEQID NO: 290) 288) GS002 Heavy GYSMS SISPGGSSTYYADSVKG YRVWHMTTGDYYSNAMDVChain (SEQ ID NO: (SEQ ID NO: 292) (SEQ ID NO: 293) 291) LightTGSSSNIGSNAVS DDNKRPS GTWDDSLSG Chain (SEQ ID NO: (SEQ ID NO: 295) (SEQID NO: 296) 294) GS003 Heavy NYSMS VISPGSGSTYYADSVKG YRVMRSPHNPYYSYGMDVChain (SEQ ID NO: (SEQ ID NO: 298) (SEQ ID NO: 299) 297) LightSGSSSNIGSNDVN SNSQRPS GSWDASLSG Chain (SEQ ID NO: (SEQ ID NO: 301) (SEQID NO: 302) 300) GS004 Heavy NYSMS AISPGSSNKYYADSVKG YRVWAHSASSSYSNAMDVChain (SEQ ID NO: (SEQ ID NO: 303) (SEQ ID NO: 304) 297) LightSGSSSNIGNNDVS SDSQRPS GSWDDSLNG Chain (SEQ ID NO: (SEQ ID NO: 306) (SEQID NO: 307) 305) GS005 Heavy NYAMS VISPDSSSKYYADSVKG AAVRRYGPSPYYYYAMDVChain (SEQ ID NO: (SEQ ID NO: 309) (SEQ ID NO: 310) 308) LightTGSSSNIGNNYVS ADSHRPS ATWDYSLSG Chain (SEQ ID NO: (SEQ ID NO: 312) (SEQID NO: 313) 311) GS006 Heavy GYSMS AISPGGGSKYYADSVRG YRVQKSAKNVYSSNGMDVChain (SEQ ID NO: (SEQ ID NO: 314) (SEQ ID NO: 315) 291) LightSGSSSNIGNNAVN YDSNRPS GAWDSSLSA Chain (SEQ ID NO: (SEQ ID NO: 317) (SEQID NO: 277) 316) GS007 Heavy NYSMS VISHDGRSKYYADSVKG FRVWKRTNAHSYANAMDVChain (SEQ ID NO: (SEQ ID NO: 318) (SEQ ID NO: 319) 297) LightTGSSSNIGSNSVS ANSNRPS GTWDASLSA Chain (SEQ ID NO: (SEQ ID NO: 321) (SEQID NO: 322) 320) GS008 Heavy NYAMS VISPDNGSTYYADSVKG ARVHCRRDQCYSYGMDVChain (SEQ ID (SEQ ID NO: 323) (SEQ ID NO: 324) NO: 308) LightTGSSSNIGSNNVT ANSQRPS GAWDSSLNG Chain (SEQ ID NO: (SEQ ID NO: 326) (SEQID NO: 327) 325) GS009 Heavy NYSMS VISPSGGNTYYADSVKG YRVKQMLNQRSYSNAMDVChain (SEQ ID NO: (SEQ ID NO: 328) (SEQ ID NO: 329) 297) LightTGSSSNIGNNYVS DDSQRPS GSWDASLSG Chain (SEQ ID NO: (SEQ ID NO: 330) (SEQID NO: 302) 311) GS010 Heavy NYSMS VISPSSGSIYYADSVKG YRVRQLKHTRSYADAMDVChain (SEQ ID NO: (SEQ ID NO: 331) (SEQ ID NO: 332) 297) LightSGSSSNIGSNAVS ANSHRPS ASWDSSLSG Chain (SEQ ID NO: (SEQ ID NO: 334) (SEQID NO: 335) 333) GS011 Heavy NYAMS GISPGGSSIYYADSVKG YAVYFLRSHGSYDYGMDVChain (SEQ ID NO: (SEQ ID NO: 336) (SEQ ID NO: 337) 308) LightTGSSSNIGSNDVT SNNQRPS GAWDYSLNA Chain (SEQ ID NO: (SEQ ID NO: 339) (SEQID NO: 340) 338) GS012 Heavy NYSMS GISPGSGSKYYADSVRG RARRFDY Chain (SEQID NO: (SEQ ID NO: 341) (SEQ ID NO: 342) 297) Light TGSSSNIGNNAVTSNSQRPS GSWDSSLNG Chain (SEQ ID NO: (SEQ ID NO: 301) (SEQ ID NO: 344)343) GS013 Heavy NYAMS SISSGGGSTYYADSVKG KKSQFDY Chain (SEQ ID NO: (SEQID NO: 345) (SEQ ID NO: 346) 308) Light SGSSSNIGSNTVN ANSQRPS DTWDYSLSGChain (SEQ ID NO: (SEQ ID NO: 326) (SEQ ID NO: 348) 347) GS014 HeavyNYAMS VISPNSGSNTYYADSVKG PRILRRRVDHSYSYAMDV Chain (SEQ ID NO: (SEQ IDNO: 349) (SEQ ID NO: 350) 308) Light TGSSSNIGNNYVS SNSHRPS GTWDYSLSGChain (SEQ ID NO: (SEQ ID NO: 351) (SEQ ID NO: 352) 311) GS015 HeavyDYSMS AISPDGGSKYYADSVKGR FRVIKLRAGWYSANGMDV Chain (SEQ ID NO: (SEQ IDNO: 354) (SEQ ID NO: 355) 353) Light SGSSSNIGNNDVS YNSKRPS GAWDDSLNGChain (SEQ ID NO: (SEQ ID NO: 357) (SEQ ID NO: 358) 356) GS016 HeavyNYAMS GIYSGNGNTYYADSVKG ALSSCPRGPCYYDDGMDV Chain (SEQ ID (SEQ ID NO:359) (SEQ ID NO: 360) NO: 308) Light SGSSSNIGSNDVT DNSKRPS GTWDASLSAChain (SEQ ID NO: (SEQ ID NO: 362) (SEQ ID NO: 322) 361) GS017 HeavyGYAM GISPGDGSTYYADSVKG VARMCQGWRCSYADGMDV Chain (SEQ ID NO: (SEQ ID NO:364) (SEQ ID NO: 365) 363) Light TGSSSNIGNNSVY SDSHRPS GTWDSSLSG Chain(SEQ ID NO: (SEQ ID NO: 367) (SEQ ID NO: 368) 366) GS018 Heavy NYAMSVISPGSGSKYYADSVKG HRVIKINRQTYYDYGMDV Chain (SEQ ID NO: (SEQ ID NO: 369)(SEQ ID NO: 370) 308) Light SGSSSNIGSNTVS SDNNRPS GAWDDSLSA Chain (SEQID NO: (SEQ ID NO: 372) (SEQ ID NO: 373) 371) GN328 Heavy TAWMDNINPDGSEKHYVDSVKG ALDY Chain (SEQ ID NO: (SEQ ID NO: 375) (SEQ ID NO:376) 374) Light RASQNINTWLA EASSLES QQYNTYS Chain (SEQ ID (SEQ ID NO:378) (SEQ ID NO: 379) NO: 377) GN337 Heavy SGGYHWN YIFNSGNTDYNPSLHRSRLLRWFDY Chain (SEQ ID NO: (SEQ ID NO: 381) (SEQ ID NO: 382) 380)Light QASQDIRKYLN DASTLET QQHDFVP Chain (SEQ ID NO: (SEQ ID NO: 384)(SEQ ID NO: 385) 383) GN414 Heavy TYGIT RITPNNGVTDYAQKFQG EIGSSSWKLLDPChain (SEQ ID NO: (SEQ ID NO: 387) (SEQ ID NO: 388) 386) LightTGNSNNVGYEGAA SDRNHNRPS SAWDSSLSE Chain (SEQ ID NO: (SEQ ID NO: 390)(SEQ ID NO: 391) 389)

1-3: Measurement of Quantitative Binding Capacity of Anti-GPC3 Antibodyto Antigen

Quantitative binding capacity (affinity) of GX102 and GX270 cloneantibodies, purified anti-GPC3 antibodies, to recombinant human GPC 3was measured using a Biacore T-200(GE Healthcare, U.S.) biosensor. Afterimmobilizing GPC3 (Cat. No. 2119-GP-050, R&D systems) purified fromHEK293 cells onto a CM5 chip (GE Healthcare) to an Rmax of 200 using anamine-carboxyl reaction, the GX102 or GX270 antibody sequentiallydiluted in a HBS-EP buffer solution (10 mM HEPES, pH7.4, 150 mM NaCl, 3mM EDTA, 0.005% surfactant P20) was flowed at a flow rate of 30 μL/minin a concentration range of 0.078 nM to 5 nM for an association time of120 seconds and a dissociation time of 1800 seconds. Dissociation of theantibody bound to GPC3 was induced by flowing 10 mM Glycine-HCl (pH1.5)at a flow rate of 30 μL/min for 30 seconds (Table 3). Affinity wasobtained as kinetic rate constants (K_(on) and K_(off)) and anequivalent dissociation constant (K_(D)) using a Biacore T-200evaluation software (Table 4).

TABLE 3 SPR Biacore T200 Chip CM5 Running Buffer HBS-EP pH 7.4 Flow rate30 μl/Min Association/Dissociation time 120 Sec/600 Sec IgG Conc.0.078~5 nM, ½ serial dilution Regeneration 10 mM Glycine-HCl pH 1.5, 30Sec

TABLE 4 K_(on) K_(off) K_(D) GX102 9.1 × 10⁶ 8.4 × 10⁻⁵ 9.2 × 10⁻¹²GX270 2.4 × 10⁶ 1.7 × 10⁻⁴ 7.0 × 10⁻¹¹

Example 2: Fluorescence Activated Cell Sorter (FACS) Analysis on Bindingof AntiGlypican 3 Antibody to Cancer Cell Expressing GPC3

In order to evaluate whether the anti-GPC3 antibody derived from thesynthetic library was selectively bound to cells expressing GPC3, anexpression amount of GPC3 was measured in a cancer cell line, andbinding of the antibody was confirmed using a FACS experiment. Table 5illustrates antibody clones used for FACS screening for cancer cellsexpressing GPC3.

TABLE 5 Source Number Clones Synthetic Library 41 GS001, GS018, GN328,GN337, GN414, GX090, GX092, GX099, GX102, GX107, GX118, GX119, GX122,GX184, GX206, GX207, GX209, GX225, GX234, GX247, GX248, GX253, GX263,GX268, GX270

2-1: Preparation of Cell Line Expressing GPC3

An expression amount of GPC3 mRNA in cells was confirmed in 8 kinds ofhepatic cancer cell lines (Huh-7, HepG2, Hep3B, SNU398, SNU475, SNU449,PLC/PRF/5, and SK-Hep1) using a real time-polymerase chain reaction(RT-PCR) method. After a TrypLE Express solution was added to the celllines cultured in a 6-well plate to detach the cell lines, a total RNAwas collected using a trizol solution. In order to amplify the GPC3mRNA, a forward primer (5′ GGA CTT GGC CAC GTT CAT G 3′) and a reverseprimer (5′ ACC TCA GCC ACA GTC AAC GG 3′) were used. As a comparisonreference for quantitative comparison, a primer set (5′ CTT CGC TCT CTGCTC CTC CT 3′, 5′ CCA GTG GAC TCC ACG ACG TA 3′) for a GAPDH mRNA wasused. After the total RNA obtained by separation using the trizolsolution was quantified using an OD quantification method, 100 ng ofcell RNA and 0.5 pM primer were put into a Maxim RT-PCR premix tube anda total volume was set to 20 μL using a nuclease-free distilled water,and a reaction was carried out at 45° C. for 30 minutes. Immediately,after an inactivation process was performed at 94° C. for 5 minutes, apolymerase chain reaction (PCR) was performed for 30 cycles of 94° C.for 30 seconds, 55° C. for 30 seconds, and 72° C. for 1 minute. In orderto complete an unreacted reaction after the last cycle, the resultantwas additionally kept at 72° C. for 3 minutes, and then, 5 μL of a5-fold concentrated agarose electrophoresis sample buffer (1.25 mg/mLBromophenol Blue, 1.25 mg/mL xylene cyanol, 30% glycerol, 25 mM Tris,pH7.6) was added thereto. After each of the analysis samples was put on1.5% agarose gel and subjected to electrophoresis at 100 volts for 15minutes, GPC3 and GPADH DNA fragments amplified by RT-PCR were examinedat a UV wavelength (FIG. 1).

Sequences of primers used for amplification of cDNA (amplicon size 758bp) encoding GPC3 were as follows.

Forward primer (sense primer): 5′GGA CTT GGC CAC GTT CAT G 3′ Reverseprimer (anti-sense primer): 5′AAC TCA GCC ACA GTC AAC GG 3′

In addition, sequences of primers used for amplification of cDNA(amplicon size 379 bp) encoding GAPDH were as follows.

Forward primer (sense primer): 5′CTT CGC TCT CTG CTC CTC CT 3′ Reverseprimer (anti-sense primer): 5′CCA GTG GAC TCC ACG ACG TA 3′

In SK-Hep1 hepatic cancer cells confirmed as a GPC3 negative cell line,a plasmid (pCMV/GPC3) having a GPC3 expression unit and Hygromycinresistant gene was delivered into cells using a jet-polyethyleneimine(PEI) transfection system (Polyplus, 101-40) (FIG. 2). After 48 hours,the cell culture solution was replaced with a culture medium containingHygromycin B (200 μg/mL). While the culture medium was replaced every 3days, 7 kinds of colonies having resistance against Hygromycin wereobtained (FIG. 3).

2-2: Analysis of Expression Amount of GPC3 in Cell Line Expressing GPC3and Tumor Cell Line

GPC3 existing on surfaces of cells was measured with respect to a cellline (SK-Hep1-GPC3) in which GPC3 was artificially expressed using aFACS experiment. After detaching cells to be analyzed, cultured in aculture dish by adding a TrypLE Express solution thereto, the detachedcells were put into a 50 mL tube and centrifuged at room temperature and2000 rpm for 3 minutes to remove a culture medium, followed by washingwith PBS once. The resultant was suspended using a FACS buffer,transferred to a round bottom tube, and centrifuged at room temperatureand 2000 rpm for 3 minutes. A supernatant was removed, and the resultantwas suitably dissolved using the FACS buffer so as to have aconcentration of 4×105 ea/mL. Then, as a FACS analysis antibody forGPC3, a mouse anti-GPC3 antibody (R&D systems) was used at 4° C., and asan isotope control group, a mouse IgG (1m, R&D systems) was used. After1 hour, the resultant was washed with the FACS buffer two times, theanti-mouse IgG antibody and PE conjugated were added thereto at anamount of 5 μL per sample and bound thereto at 4° C. for 30 minutes.After collecting cells by centrifugation at 2000 rpm for 3 minutes, thecells were re-suspended by adding a fixation buffer (500 μL), andmeasured using a FACS calibur (FIG. 4).

As a result, it was confirmed that GPC3 was positive in 8 kinds ofclones (#2, #3, #5, #6, #7, #8, #9, and #Pool), and in the SK-Hep1 cellline, a mother cell line, GPC3 was negative, as illustrated in FIG. 4.

2-3: Analysis of Selective Binding of Anti-GPC3 Antibody to Cell LineExpressing GPC3

Whether or not anti-GPC3 antibody selectively binds to a cell line(SK-Hep1-GPC3) in which GPC3 was artificially over-expressed wasmeasured using a FACS experiment. After detaching SK-Hep1-GPC3 cells byadding a TrypLE Express solution thereto, the detached SK-Hep1-GPC3cells were put into a 50 mL tube and centrifuged at room temperature and2000 rpm for 3 minutes to remove a culture medium, followed by washingwith PBS once. The resultant was suspended using a FACS buffer,transferred to a round bottom tube, and centrifuged at room temperatureand 2000 rpm for 3 minutes. A supernatant was removed, and the resultantwas suitably dissolved using the FACS buffer so as to have aconcentration of 4×10⁵ ea/mL. Then, a candidate antibody (1 μg) wasadded thereto at 4° C., and in an isotope control group, a human IgG(1m, Sigma) was added instead of the candidate antibody. After 1 hour,the resultant was washed with the FACS buffer two times, a goatanti-human IgG antibody and FITC conjugated were added thereto at anamount of 1 μL per sample and bound thereto at 4° C. for 30 minutes.After collecting cells by centrifugation at 2000 rpm for 3 minutes, thecells were re-suspended by adding a fixation buffer (500 μL), andmeasured using a FACS calibur (FIG. 5).

In order to compare binding capacity between the analyzed candidateantibodies, a reference antibody was set in each analysis, and thebinding capacity was compared based on mean fluorescent intensity (MFI)(FIG. 6). Since the SK-Hep1-GPC3 cells were cell lines in which GPC3 wasartificially over-expressed, it is impossible to predicate that theSK-Hep1-GPC3 cells maintained an original GPC3 structure as it is.Therefore, whether or not the anti-GPC3 antibody was selectively boundto cells even in hepatic cancer cells (HepG2) in which GPC3 wasoriginally expressed was compared and analyzed by the same method asdescribed above (FIGS. 7 and 8). As a result, it was confirmed that theantibodies having excellent binding capacity in both of the two celllines (SK-Hep1-GPC3 and HepG2) were GX102, GX270, and GS012 clone.

In order to confirm whether or not the GX102, GX270, and GS012 cloneantibodies having excellent binding capacity to GPC3 selectively bind tocell lines in proportion to an expression level of GPC3 in tumor cells,FACS binding was measured in a total of seven kinds of hepatic cancercell lines (SK-Hep1, PLC/PRF/5, SNU398, Hep3B, Huh7, HepG2, andSK-Hep1-GPC3 #9) and one kind of normal hepatic cancer cell line (CHANG)(FIG. 9). The results were represented and compared by positive signsdepending on an overlapping profile of a FACS histogram with that in anisotope negative control group (Table 6 and FIG. 10).

As a result, GX102 and GX270 candidate antibodies were selectively boundto the hepatic cancer cell lines in proportion to the expression levelof GPC3 as illustrated in Table 6 and FIGS. 9 and 10. On the contrary,it was confirmed that the GS012 antibody was non-specifically bound toall of the hepatic cancer cell lines regardless of the expression levelof GPC3.

TABLE 6 GPC3Ab SK-Hep1 PLC/PRF/5 CHANG SNU398 Hep3B Huh7 HepG2SK-Hep1-GPC3 #9 GPCSexp. − − − −/+ + + + + + + +GS012 + + + + + + + + + + + + + + + + + + + + + + GX102 − − −− + + + + + + + GX270 − − + − − + + + +

INDUSTRIAL APPLICABILITY

An antibody specifically binding to glypican 3 according to the presentinvention may be effectively used to treat cancer or tumor,particularly, hepatocellular carcinoma due to high affinity andspecificity to glypican 3.

Although the present invention has been described in detail based onparticular features thereof, it is obvious to those skilled in the artthat these specific technologies are merely preferable embodiments andthus the scope of the present invention is not limited to theembodiments. Therefore, the substantial scope of the present inventionis defined by the accompanying claims and equivalent thereof.

Sequence Listing Free Text

Attached electronic file.

1. An anti-glypican 3 antibody comprising a heavy chain variable regioncomprising the following heavy chain CDRs: a heavy chain CDR1 comprisingan amino acid sequence of SEQ ID NO: 133, 143, 149, 159, 169, 174, 184,199, 262, 268, 273, 281, 285, 291, 297, 308, 353, 363, 374, 380, or 386;a heavy chain CDR2 comprising an amino acid sequence of SEQ ID NO:134,144, 150, 160, 170, 175, 185, 200, 231, 263, 266, 269, 274, 282, 286,292, 298, 303, 309, 314, 318, 323, 328, 331, 336, 341, 345, 349, 354,359, 364, 369, 375, 381, or 387; and a heavy chain CDR3 comprising anamino acid sequence of SEQ ID NO: 135, 139, 145, 151, 155, 161, 165,171, 176, 180, 186, 189, 194, 195, 201, 205, 207, 210, 214, 218, 222,227, 232, 237, 239, 243, 247, 250, 254, 258, 259, 264, 267, 270, 275,278, 279, 283, 287, 293, 299, 304, 310, 315, 319, 324, 329, 332, 337,342, 346, 350, 355, 360, 365, 370, 376, 382, or 388, or a light chainvariable region comprising the following light chain CDRs: a light chainCDR1 comprising an amino acid sequence of SEQ ID NO: 136, 140, 146, 152,156, 162, 166, 172, 177, 181, 187, 190, 193, 196, 202, 208, 211, 215,219, 223, 224, 228, 233, 240, 244, 251, 255, 260, 265, 271, 288, 294,300, 305, 311, 316, 320, 325, 333, 338, 343, 347, 356, 361, 366, 371,377, 383, or 389; a light chain CDR2 comprising an amino acid sequenceof SEQ ID NO: 137, 141, 147, 153, 157, 163, 167, 178, 182, 191, 197,203, 209, 212, 216, 220, 225, 229, 234, 236, 241, 245, 252, 256, 276,284, 289, 295, 301, 306, 312, 317, 321, 326, 330, 33339, 351, 357, 362,367, 372, 378, 384, 390, or 392, and a light chain CDR3 comprising anamino acid sequence of SEQ ID NO. 138, 142, 148, 154, 158, 164, 168,173, 179, 183, 188, 192, 198, 204, 206, 213, 217, 221, 226, 230, 235,238, 242, 246, 248, 249, 253, 257, 261, 272, 277, 280, 290, 296, 302,307, 313, 322, 327, 335, 340, 344, 348, 352, 358, 368, 373, 379, 385, or391.
 2. (canceled)
 3. The anti-glypican 3 antibody according to claim 1,comprising a heavy chain variable region comprising a sequence with ahomology of 80% or more with an amino acid sequence of SEQ ID NO: 1, 3,5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41,43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77,79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109,111, 113, 115, 117, 119, 121, 123, 125, 127, 129, or
 131. 4. Theanti-glypican 3 antibody according to claim 1, comprising a light chainvariable region comprising a sequence with a homology of 80% or morewith an amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16,18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52,54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88,90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118,120, 122, 124, 126, 128, 130, or
 132. 5. A conjugate comprising theanti-glypican 3 antibody according to claim 1 and a drug.
 6. Theconjugate according to claim 5, wherein the drug is an agent fortreating cancer.
 7. The conjugate according to claim 6, wherein the drugis at least one selected from the group consisting of microtubulininhibitor, a mitosis inhibitor, a topoisomerase inhibitor, a DNAintercalator, a protein toxin performing an enzymatic function,radioisotopes (radionuclides), miRNA, shRNA and siRNA.
 8. The conjugateaccording to claim 6, wherein the drug is at least one selected from thegroup consisting of maytansinoid, auristatin, dolastatin, trichothecene,CC1065 (Cytotoxic compound), calicheamicin and other enediyneantibiotics, taxane, anthracycline, methotrexate, adriamycin, vindesine,vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin,melphalan, mitomycin C, chlorambucil, daunorubicin, daunomycin,etoposide, teniposide, carminomycin, aminopterin, dactinomycin,mitomycins, bleomycins, esperamicins, 5-fluorouracil, melphalan, othernitrogen mustards and stereoisomers, isosteres, analogues, orderivatives thereof, cis-platinum and cis-platinum analogues, enzymesand fragments thereof corresponding to other intercalating agents,nucleolytic enzymes, antibiotics, and toxins (enzymatically activetoxins or small molecule toxins of bacterial, fungal, plant or animalorigin), various antitumor or anticancer agents such as cisplatin,CPT-11, doxorubicin, paclitaxel and docetaxel.
 9. A bispecific antibodycomprising the anti-glypican 3 antibody according to claim
 1. 10. Thebispecific antibody according to claim 9, comprising the anti-glypican 3antibody and an antibody having binding ability to an immune effectorcell-specific target molecule.
 11. The bispecific antibody according toclaim 10, wherein the immune effector cell-specific target molecule isselected from the group consisting of TCR/CD3, CD16(FcγRIIIa) CD44,Cd56, CD69, CD64(FcγRI), CD89 and CD11b/CD18(CR3).
 12. A method fortreating cancer comprising administering the antibody according toclaim
 1. 13. The method for treating cancer according to claim 12,wherein the cancer is selected from the group consisting of hepaticcancer, hepatocellular carcinoma, gastric cancer, breast cancer, lungcancer, ovarian cancer, bronchial cancer, nasopharyngeal cancer, larynxcancer, pancreatic cancer, bladder cancer, colorectal cancer, coloncancer, uterine cervical cancer, brain cancer, prostate cancer, bonecancer, skin cancer, thyroid cancer, parathyroid cancer, kidney cancer,esophageal cancer, biliary tract cancer, testis cancer, rectal cancer,head and neck cancer, cervical spinal cancer, ureteral cancer,osteosarcoma, neuroblastoma, melanoma, fibrosarcoma, rhabdomyosarcoma,astrocytoma, neuroblastoma, and glioma. Most preferably, the cancercapable of being treated with the composition according to the presentinvention is hepatic cancer or hepatocellular carcinoma.
 14. The methodfor treating cancer according to claim 13, wherein the cancer is hepaticcancer, hepatocellular carcinoma, breast cancer, ovarian cancer,colorectal cancer, skin cancer, biliary tract cancer or lung cancer. 15.A polynucleotide encoding the anti-glypican 3 antibody according to aclaim 1 to
 4. 16. A recombinant vector comprising the polynucleotideaccording to claim
 15. 17. A host cell transformed with the recombinantvector according to claim
 16. 18. The host cell according to claim 17,the cell is selected from the group consisting of animal cell, plantcell, yeast, E. coli, and insect cell.
 19. The host cell according toclaim 17, the cell is selected from the group consisting of monkeykidney cells 7 (COS7), NSO cells, SP2/O, Chinese hamster ovary (CHO)cells, W138, baby hamster kidney (BHK) cells, Madin-Darby canine kidney(MDCK) cells, myeloma cell lines, HuT 78 cells, HEK293 cells,Escherichia coli, Bacillus subtilis, Streptomyces sp., Pseudomonas sp.,Proteus mirabilis, Staphylococcus sp. Aspergillus sp., Pichiapastoris,Saccharomyces cerevisiae, Schizosaccharomyces sp., and Neurosporacrassa.
 20. A method of preparing an anti-glypican 3 antibody comprisingculturing a host cell according to claim
 17. 21. A compositioncomprising the anti-glypican 3 antibody according to claim 1.